Inhalable Antimicrobial Particles and Methods of Making the Same

ABSTRACT

The present invention relates in part to novel drug delivery particles comprising an anionic polymer matrix and a cationic polymer, wherein the anionic polymer matrix and cationic polymer together form drug delivery particles bound by electrostatic interactions and wherein the drug delivery particles comprise at least one biologically active agent. The invention also relates in part to a method of treating a mycobacterial infection using said drug delivery particles, and a method of making said drug delivery particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This PCT application claims priority to U.S. Provisional PatentApplication Ser. No. 62/515,019, filed Jun. 5, 2017, the entire contentof which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Despite effective treatments for tuberculosis (TB), it remains a leadingcause of morbiditiy and mortality around the world. Presently, standardTB treatment regiments require patients to complete a six-month courseof a multi-drug cocktail taken daily under the direct observation of ahealthcare worker. This burden ultimately leads to inappropriate druguse and early termination of treatment, collectively contributing to thewidespread emergence of multi drug-resistant TB (MDR-TB) and extensivelydrug-resistant TB (XDR-TB) strains. Although TB vaccines are availableand provide limited defense to young children, they are all butineffective in preventing highly contagious adult pulmonary TB.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTb), remainsone of the leading causes of mortality worldwide, afflicting more than 9million new people per year and causing more than 1.5 million deaths(Zumla, et al., Nat. Rev. Drug Discov. 2013, 12 (5), 388-404; Horsburgh,et al., N. Engl. J. Med. 2015, 373 (22), 2149-2160). Importantly, anestimated 2 billion individuals are infected with MTb in an asymptomaticlatent stage, and are at risk of re-emergence of the disease. Despitethe prevalence of TB infection, drug-susceptible TB can be effectivelytreated through directly observed therapy (DOT) in which patientsreceive a multi-drug oral cocktail (rifampicin, isoniazid, pyrazinamideand ethambutol) that is taken daily for 6 months. If completed thisregimen leads to cure rates that are >95%. However, several issuescomplicate current therapy, including drug intolerance and toxicity,drug-drug interactions and the burdensome length of treatment requiredto achieve a re-lapse free cure (Chan and Iseman, Curr. Opin. Infect.Dis. 2008, 21 (6), 587-595). As a result, patients are routinelynon-compliant, either taking drugs inappropriately outside of DOT orterminating treatment early (Munro, et al., PLoS Med. 2007, 4 (7),e238). These challenges have contributed to the widespread emergence ofmulti drug-resistant TB (MDR-TB) and extensively drug-resistant TB(XDR-TB) strains, that require ‘individualized’ treatments using second-and third-line antibiotics that are more expensive, have significantlyincreased toxicities and must be administered for up to 24 months(Gandhi, et al., The Lancet 2010, 375 (9728), 1830-1843; Keshavjee andFarmer, N. Engl. J. Med. 2012, 367 (10), 931-936).

To complicate matters, MTb can reside within niche environments in thelung where they are protected from the action of antibiotics(Ramakrishnan, Nat. Rev. Immunol. 2012, 12 (5), 352-366). For example,mycobacteria colonizing the alveolar epithelium are phagocytosed by lungmacrophages and trafficked to the phagolysosome. However, MTb hasevolved mechanisms to inhibit phagosome maturation and avoid hydrolyticdestruction, ultimately allowing them to replicate unperturbed in theinfected host cell (Russell, et al., Nat.

Immunol. 2009, 10 (9), 943-948). In an attempt to contain the infection,the immune system sequesters MTb and infected macrophages withingranulomas that, counter-productively, limits the diffusion ofantibiotics into the tissue and creates an anaerobic environment thatrenders MTb dormant and phenotypically resistant to standard drugs(Horsburgh, et al., N. Engl. J. Med. 2015, 373 (22), 2149-2160). Thesechallenges, along with the spread of drug-resistant disease, has createda renewed urgency for new anti-TB therapeutic candidates that can 1)elicit their activity on targets distinct from conventional antibiotics,and thus are effective against drug-resistant bacteria, 2) kill bothproliferative and non-replicating dormant MTb, and 3) are sufficientlypotent to shorten the course of treatment. Conversely, current TB drugsare still effective but require more efficient delivery strategies to beoperational (Griffiths, et al., Nat. Rev. Microbiol. 2010, 8 (11),827-834). Thus, carrier systems that can co-deliver new classes ofanti-TB agents with standard antibiotics, while providing long-term drugrelease, represents an effective and compliant strategy in the treatmentof drug-susceptible and -resistant TB.

Replacing the oral administration of free antibiotics with inhalabledrug-loaded microparticles is an attractive strategy for TB therapy asit increases the local concentration of drug at the infection site,thereby enhancing potency and minimizing off-target toxicity, whilecontrollably releasing the cargo to afford reduced frequency of dosing(Griffiths, et al., Nat. Rev. Microbiol. 2010, 8 (11), 827-834).Traditional approaches have utilized chemically-crosslinkedpoly(lactic-co-glycolic acid) (PLGA) polymer microspheres, in whichantibiotics are encapsulated within the porous particle matrix. Cell-and animal-based studies show that PLGA particles prolongtherapeutically relevant concentrations of loaded antibiotics within theplasma, and allow for preferential accumulation of drug within infectedmacrophages (Sosnik, et al., Adv. Drug Delivery Rev. 2010, 62 (4),547-559; Hirota, et al., J. Controlled Release 2010, 142 (3), 339-346;O'Hara and Hickey, Pharm. Res. 2000, 17 (8), 955-961; Suarez, et al.,Pharm. Res. 2001, 18 (9), 1315-1319). Despite the popularity of thisapproach it is not without significant challenges. The organic solventsand chemical cross-linkers used to synthesize PLGA microspheres aretoxic and can induce severe allergic reactions in patients, thuslimiting their clinical utility (Griffiths, et al., Nat. Rev. Microbiol.2010, 8 (11), 827-834).⁹ More importantly, these technologies delivertraditional TB antibiotics and thus do not effectively circumvent drugresistance.

Modern high throughput screening campaigns have identified an abundanceof biochemical probes and therapeutic candidates with unprecedentedspecificity and potency. These agents, if successfully translated intothe clinic, could transform strategies in precision medicine and lead tothe design of highly selective antimicrobials. Yet, many potentiallyefficacious lead molecules are abandoned due to poor water solubility,low bioavailability, rapid systemic clearance and off-targetbiodistribution to healthy tissues leading to dose-limiting adverseevents. Even many clinically approved pharmaceutics must be formulatedwith toxic adjuvants and/or excipients that can compound the sideeffects of the active agent.

Incorporation of diagnostic or therapeutic cargo into bioresponsivenanomaterials, such as a polymer or lipid-based nanoparticle, canaddress these challenges by improving the pharmacologic and therapeuticproperties of loaded agents when parenterally administered. Chemicalligation or physical encapsulation of biomolecular cargo within anano-carrier matrix leads to enhanced aqueous solubility, improved serumstability and affords preferential localization to diseased tissuesthrough size-dependent passive targeting. Considerable efforts are nowbeing made to develop bioresponsive nano-scale vehicles to improve thetransport of sensitive protein and nucleic-acid based agents for genomeediting, biotherapy and biosensing applications. While a number of‘smart’ delivery systems have been designed to address this need,successful translation of these platforms into the clinic has remainedelusive due to their significant chemical complexity, substantial costto scale and toxicity of the matrix constituents upon carrierdegradation in physiologic environments.

There is need in the art for novel drug delivery formulations. Thepresent invention satisfies this unmet need.

SUMMARY OF THE INVENTION

The present invention relates in part to a plurality of drug deliveryparticles, comprising an anionic polymer matrix; and a cationic polymer;wherein the anionic polymer matrix and cationic polymer together formdrug delivery particles bound by electrostatic interactions; and whereinthe drug delivery particles comprise at least one biologically activeagent. In one embodiment, the anionic polymer matrix comprises ananionic polymer selected from the group consisting of alginic acid,arabic acid, polygalacturonic acid, poly(glucuronic acid), hyaluronicacid, heparin, N-acetyl heparin, carboxymethylcellulose, chondroitinsulfate, chondroitin sulfate B, chitin, O- or N-sulfochitosan,CM-dextran, dextran sulfate, and pectin. In one embodiment, the anionicpolymer matrix comprises hyaluronic acid. In one embodiment, thecationic polymer is a polypeptide. In one embodiment, the cationicpeptide is poly-L-lysine. In one embodiment, the cationic peptidecomprises a sequence selected from the group consisting of WKWLKKWIK,ILRWKWRWWRWRR, KRWWKWWRR, and RRWWRWVVW. In one embodiment, the zetapotential of the particles is negative. In one embodiment, the at leastone biologically active agent is selected from the group consisting ofan antimycobacterial agent, an antimicrobial agent, an antiviral agent,an anticancer agent, and a biologic. In one embodiment, the at least onebiologically active agent is selected from the group consisting ofrifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin,4-chloro-N-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)benzamide,vancomycin, and doxorubicin. In one embodiment, the drug deliveryparticles further comprise at least one pharmaceutically acceptablecarrier.

The present invention also relates in part to a dry powder formulationcomprising the particles of the invention, and a method of treating amycobacterial infection in a subject in need thereof, the methodcomprising the step of administering to the subject a formulationcomprising the particles of the invention.

The present invention also relates in part to A method for themanufacture of drug delivery particles, the method comprising the stepsof: providing a sample solution comprising at least one anionic polymer;providing a bath solution comprising at least one cationic polymer;electrospraying the sample solution into the bath solution to form adrug delivery particle solution; and isolating a plurality of drugdelivery particles from the drug delivery particle solution; wherein atleast one of the sample solution, the bath solution, or the solution ofdrug delivery particles further comprises at least one biologicallyactive agent. In one embodiment, the step of isolating a plurality ofdrug delivery particles from the drug delivery particle solutioncomprises the step of centrifuging the drug delivery particle solutionand removing the supernatant. In one embodiment, the step of isolating aplurality of drug delivery particles from the drug delivery particlesolution further comprises the step of lyophilizing the drug deliveryparticles. In one embodiment, the step of electrospraying the samplesolution into the bath solution to form a drug delivery particlesolution further comprises the step of incubating the drug deliverysolution for at least one hour at least 37° C. In one embodiment, thebath solution comprises the biologically active agent. In oneembodiment, the at least one anionic polymer comprises hyaluronic acid.In one embodiment, the at least one cationic polymer comprisespoly-L-lysine or an antimicrobial polypeptide. In one embodiment, theelectrospray voltage is between about 10 kV and about 50 kV.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of various embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings illustrative embodiments. It should beunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities of the embodiments shown in thedrawings.

FIG. 1 is a flowchart of an exemplary method for the production of anantimicrobial formulation.

FIG. 2 depicts the delivery of an exemplary anti-TB microgel compositionto the lungs. Positively charged AMPs (orange) are complexed with FDAapproved hyaluronic acid (grey) to form biodegradable microgels in highyield and low cost. Antibiotics loaded into the particle core (purple)are slowly released via degradation of the carbohydrate matrix bybacterial enzymes, affording sustained and long-term TB therapy.

FIG. 3 depicts three possible mechanisms of combinatorial TB therapy byexemplary drug-loaded AMP microgels. Left, contact of drug-sensitive and-resistant MTb (green) with AMPs (orange) displayed from microgelparticles (tan) leads to rapid bacteriolytic activity. Middle, particledegradation by MTb-secreted enzymes affords controlled and sustainedrelease of encapsulated antibiotics (purple) to infected lung tissue.Right, phagocytosis and degradation of microgels by infected macrophages(pink) affords intracellular release of AMPs and antibiotics tosterilize the infected host cells. Subsequent containment of infectedmacrophages within granulomas may allow for trafficking of AMPs andantibiotics to non-replicating persister cells.

FIG. 4, comprising FIGS. 4A-4D, depicts the application and results ofan exemplary electrospray process. FIG. 4A depicts an exemplary AMPmicrogel synthesis via electrospray ionization. Here, an aqueoussolution of anionic HA (tan) is infused through a charged capillary (24kV) causing it to spray as a fine mist. Collection of HA droplets into abath of cationic AMPs leads to rapid electrostatic cross-linking andassembly of microgels. Antibiotics present during microgel formationbecome loaded within the particle core. FIG. 4B is a plot showing theparticle size of the microgels in water, as measured by dynamic lightscattering. FIG. 4C is a plot of the surface charge of TB1 microgels inwater, as measured by zeta potential analysis. FIG. 4D is a scanningelectron micrograph of exemplary TB1 particles (scale bar=1 μm).

FIG. 5, comprising FIGS. 5A and 5B, depicts biological activities ofexemplary antimicrobial compositions. FIG. 5A is a plot of cellviability; the novel trans-translation inhibitor KKL-35 was added atindicated concentrations to cultures of MTb and cell viability measuredvia plating assays. FIG. 5B is a plot of the viability of MTb under the“Wayne model.” Non-replicating but viable MTb cultures were generated bygradual consumption of oxygen inside sealed glass tubes. KKL-35 wasinjected anaerobically and cultures stirred for 3 days. Viable bacteriawere enumerated by plating. The dashed line shows CFU/mL at the time ofaddition.

FIG. 6 is a photograph showing exemplary microgels prepared using themethods of the present invention.

FIG. 7, comprising FIGS. 7A and 7B, demonstrates the biocompatibilityand bioavailability of exemplary antimicrobial compositions. FIG. 7A isa plot comparing the activity of non-drug-loaded microgels againstgram-negative bacteria M. smeg to the activity against mammalian lungepithelial cell controls. FIG. 7B is a plot showing the controlledrelease of three exemplary cargos from model particle formulations.

FIG. 8 depicts the production of exemplary drug delivery particles byelectrospray ionization. An aqueous solution of anionic HA (orange) isinfused through an electrically-charged capillary, causing it to sprayas nano-scale droplets. Contact of HA nanodroplets with ϵ-poly-L-lysine(PLL; purple) in the bath solution leads to electrostatic assembly ofnanogel particles. Therapeutic agents or biochemical sensors(green/white) present during nanogel assembly become physicallyentrapped within the particle network.

FIG. 9, comprising FIGS. 9A-9D, depicts physical characteristics ofexemplary drug delivery particles. FIG. 9A is a plot of particle size asmeasured using dynamic light scattering (DLS). FIG. 9B is an image ofthe electrospray bath solution before (left) and immediately after(right) synthesis of exemplary drug delivery particles. The rapid changein solution turbidity illustrates the high yield production ofparticles, which remain colloidally stable. FIG. 9C is a plot of a Zetapotential analysis of exemplary drug delivery particles. The negativesurface charge suggests a core-shell particle architecture in which ananionic HA corona surrounds a cationic PLL core (inset). FIG. 9D is aplot of particle diameter as a function of N:P ratio for exemplary drugdelivery particles of N:P ratio of 1:1 to 15:1.

FIG. 10, comprising FIGS. 10A-10E, depicts physicochemicalcharacteristics of exemplary drug delivery particles. FIG. 10A is a plotof particle size, as measured by DLS, under various applied electrosprayvoltages. FIG. 10B is a plot of particle size at various N:P ratios, asmeasured by DLS. FIG. 10C is a plot depicting the zeta potential ofparticles prepared at N:P ratios of 5 and 10. FIG. 10D is a chartshowing the stability of particle size when stored in DI water. FIG. 10Eis a plot of particle size following loading of the model protein GFP(NG_(GFP)), the chemotherapeutic agent DOX (NG_(DOX)), or the antibioticVAN (NG_(VAN)), as determined by DLS. Unloaded NG shown for reference.

FIG. 11, comprising FIGS. 11-11, depicts the effects of prolongedexposure of exemplary drug delivery particles to physiologic media. FIG.11A is a plot of relative nanogel swelling in physiologic media at N:Pratios of 1:1 to 15:1. Dotted lines signify disruption of particleintegrity as indicated by loss of DLS signal. Note, N:P=1 nanogelsrapidly degrade between 0 and 0.5 hours. FIG. 11B depicts scanningelectron micrographs (SEMs) of exemplary drug delivery particlesnanogels before (top) and after (bottom) 18 hours of swelling; (scalebar=500 um) FIG. 11C is a plot of time to particle degradation as afunction of N:P ratio.

FIG. 12, comprising FIGS. 12A-12D, depicts the utility of exemplary drugdelivery particles for drug delivery applications. FIG. 12A is aschematic showing the various loading methods available for theencapsulation of molecular cargoes within exemplary drug deliveryparticles. Vancomycin (VAN, blue) is suspended in the HA samplesolution, while Green fluorescent protein (GFP, green) is present in thebath solution, leading to their encapsulation during nanogel assembly.Doxorubicin (DOX, red) is incubated with pre-formed drug deliveryparticles, leading to its adsorption within the particle amphiphilicmatrix. FIG. 12B is a plot of the fraction of cargo released as afunction of time. FIG. 12C is a plot of the zeta potential analysis ofun-loaded nanogels (NG) compared to formulations encapsulating thevarious molecular cargoes. FIG. 12D is an illustration of molecularcargo localization of GFP (green), VAN (blue) or DOX (red) withinexemplary drug delivery particles.

FIG. 13, comprising FIGS. 13A-13C, depicts the intake of GFP fromGFP-loaded drug delivery particles by cancer cells. FIG. 13A showsmerged confocal microscopy images of Hoechst (blue) and GFP (green)fluorescent channels for A549 lung carcinoma cells treated with freeGFP, or GFP-loaded nanogels without (NG_(GFP)) and with co-incubation ofexcess HA (NG_(GFP)+HA). (60x magnification; scale bar=10 μm). FIG. 13Bis a chart of the quantitation of average GFP fluorescence per cell foreach treatment condition (n=15; p<0.01). FIG. 13C depicts fluorescentconfocal microscopy images of A549 cells treated with free GFP orNG_(GFP), and co-stained with the endosomal marker texas-red labeledtransferrin (TransferrinTR). Individual fluorescence channels and mergedimages shown (60× magnification; scale bar=10 μm).

FIG. 14, comprising FIGS. 14A and 14B, depicts the results of cytoxicitystudies against potential therapeutic targets. FIG. 14A depicts theantibacterial effect of non-drug-loaded drug delivery particles againstthe gram-negative pathogen M smeg. compared to mammalian lung epithelialcell controls (A549). FIG. 14B shows the cytotoxicity of free DOX,DOX-loaded nanogels (NG_(DOX)) or the empty nanogel carrier (NG) againstA549 lung carcinoma cells (chart and table) and the multi-drug resistantNCI/ADR-RES ovarian cancer cell line (table only). Table results areshown as the IC₅₀ of DOX, or the equivalent concentration of drug loadedinto nanogels, as well as the corresponding amount of the carrier(represented as DOX|NG carrier). NA=not applicable. *indicates maximumconcentration tested.

FIG. 15 shows the biocompatibility of exemplary drug delivery particles.FIG. 15A is a plot of the viability of human umbilical vein endothelialcells (HUVEC) as a function of drug delivery particle concentration.FIG. 15 B is a plot of bovine red blood cell hemolysis percentagefollowing a 24 hour incubation with increasing concentrations of theempty nanogel carrier. TX=Triton X-100 positive control.

DETAILED DESCRIPTION

It is to be understood that the Figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clear understanding of the present invention, while eliminating,for the purpose of clarity, many other elements found in antimicrobialcompositions and methods of making. Those of ordinary skill in the artmay recognize that other elements and/or steps are desirable and/orrequired in implementing the present invention. However, because suchelements and steps are well known in the art, and because they do notfacilitate a better understanding of the present invention, a discussionof such elements and steps is not provided herein. The disclosure hereinis directed to all such variations and modifications to such elementsand methods known to those skilled in the art.

As used herein, each of the following terms has the meaning associatedwith it in this section. Unless defined otherwise, all technical andscientific terms used herein generally have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a sign orsymptom of the disease or disorder, the frequency with which such a signor symptom is experienced by a patient, or both, is reduced.

An “effective amount” or “therapeutically effective amount” of acompound is that amount of compound which is sufficient to provide abeneficial effect to the subject to which the compound is administered.An “effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human. As used herein, a subject is preferably a mammal such as anon-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and aprimate (e.g., monkey and human), most preferably a human.

“Parenteral” administration of a composition includes, e.g.,subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, “treating a disease or disorder” means reducing thefrequency with which a symptom of the disease or disorder is experiencedby a patient. Disease and disorder are used interchangeably herein.

The phrase “therapeutically effective amount,” as used herein, refers toan amount that is sufficient or effective to prevent or treat (delay orprevent the onset of, prevent the progression of, inhibit, decrease orreverse) a disease or condition, including alleviating symptoms of suchdiseases.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

As used herein, the term “attached to” refers to attaching two chemicalgroups through a chemical bond, for example a covalent bond or anon-covalent bond.

As used herein, the terms “amide,” “amide group,” or “amido group,”employed alone or in combination with other terms, means, unlessotherwise stated, a chemical group containing one or more amino groups.In one example, the amide group is represented by structure of—C(O)NR_(a)R_(b), wherein the carbon atom may optionally be substitutedwith sulfate or phosphate atom; and wherein, in some embodiments of theinvention, R_(a) and R_(b) are hydrogen.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent depending on thecontext in which it is used. As used herein when referring to ameasurable value such as an amount, a temporal duration, and the like,the term “about” is meant to encompass variations of ±20% or ±10%, morepreferably ±5%, even more preferably ±1%, and still more preferably±0.1% from the specified value, as such variations are appropriate toperform the disclosed methods.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Drug Delivery Particles

According to one aspect, the present invention relates to a plurality ofdrug delivery particles comprising an anionic polymer matrix; and acationic polymer; wherein the anionic polymer matrix and cationicpolymer together form drug delivery particles bound by electrostaticinteractions; and wherein the drug delivery particles comprise at leastone biologically active agent.

The anionic polymer matrix comprises any polymer having an overallnegative charge. Exemplary polymers include, but are not limited to,synthetic polymers such as polyacrylates, poly(4-styrenesulfonate),poly(vinyl sulfate), and poly(vinylphosphonic acid),poly(vinylphosphate), polymetaphosphate; polypeptides having an overallnegative charge and comprising a plurality of amino acids that arenegatively charged at physiological conditions, such as, but not limitedto, poly(glutamic acid), poly(γ-glutamic acid), poly(aspartic acid),poly(β-aspartic acid), and copolymers and block copolymers thereof;carbohydrate biopolymers such as alginic acid, arabic acid,polygalacturonic acid, poly(glucuronic acid), hyaluronic acid, heparin,N-acetyl heparin, carboxymethylcellulose, chondroitin sulfate,chondroitin sulfate B, chitin, O- or N-sulfochitosan, CM-dextran,dextran sulfate, pectin, ribonucleic acid (RNA), deoxyribonucleic acid(DNA); lignins and lignin-derived polymers; and combinations,co-polymers, and block-copolymers thereof. In some embodiments, theanionic polymer is a copolymer or a block copolymer. In someembodiments, the copolymer or block copolymer further comprises at leastone neutral monomer. In some embodiments, the copolymer or blockcopolymer further comprises at least one cationic monomer. In oneembodiment, the anionic polymer comprises hyaluronic acid. In oneembodiment, the anionic polymer comprises alginic acid. In oneembodiment, the anionic polymer comprises a polypeptide having anoverall negative charge. In one embodiment, the anionic polymercomprises polygalacturonic acid. In one embodiment, the anionic polymercomprises polyglucoronic acid. One of ordinary skill in the art wouldappreciate that the overall negative charge refers to thebackbone/framework of the polymer, and that the polymer necessarilycomprises positively-charged counterions, such as but not limited to,lithium, sodium, potassium, or ammonium ions.

There is no particular limit to the size or length of the anionicpolymer. In one embodiment, the anionic polymer has an average molecularweight between about 1 kDa and about 1,000 kDa. In one embodiment, theanionic polymer has an average molecular weight of about 1 kDa. In oneembodiment, the anionic polymer has an average molecular weight of about10 kDa. In one embodiment, the anionic polymer has an average molecularweight of about 100 kDa. In one embodiment, the anionic polymer has anaverage molecular weight of about 1,000 kDa.

The cationic polymer can be any polymer having an overall positivecharge. One of ordinary skill in the art would appreciate that theoverall positive charge refers to the backbone/framework of the polymer,and that the polymer necessarily comprises negatively-chargedcounterions, such as but not limited to, acetate, fluoride, chloride,bromide, and iodide anions. Exemplary cationic polymers include, but arenot limited to, quaternary ammonium-containing synthetic polymers suchas poly(methacryloxyethyldimethylbenzylammonium chloride)(poly(DMAEM.BzCl)), poly(methacryloxyethyldiethylmethylammoniumchloride) (poly(DEAEM.MeCl)),poly(acrylamido(2-methylbutyl)trimethylammonium chloride)(poly(AMBTAC)), poly(methacryloxyethyltrimethylammonium chloride)(poly(MOTAC.MeCl)), poly(acryloxyethyltrimethylammonium chloride)(poly(AETAC.MeCl)), poly(acrylamidopropyltrimethylammonium chloride)(poly(APTAC)), poly(methacrylamidopropyltrimethylammonium chloride)(poly(MAPTAC)), poly(methyloyloxyethyltrimethylammonium chloride)(poly(METAC)), poly(methyloyloxyethyltrimethylammonium methyl sulfate)(polyMETAMS), poly(acryloyloxyethyltrimethylammonium chloride(poly(AETAC)), poly(2-methacryloxyethyltrimethylammonium chloride)(poly(MADQUAT)), poly(dimethyldiallylammonium chloride) (poly(DMDAAC)),poly(triallylmethylammonium chloride) (poly(TAMAC)), poly(allylaminehydrochloride), polybrene, polyethyleneimine (PEI), ionene polymers,poly(N-vinylimidazole), poly(N′-alkyl-N-vinylimidazoliums),polyquaternium-1 through -47, DEAE-dextran, and combinations,copolymers, and block copolymers thereof.

In one embodiment, the cationic polymer comprises a polypeptide. In oneembodiment, the polypeptide has an overall positive charge. Suchpolypeptides comprise a plurality of amino acids that are positivelycharged at physiological conditions, including homopeptides such as, butnot limited to, α- or ϵ-poly-L-lysine, poly-L-histidine, α- orδ-poly-L-ornithine, poly-L-arginine; polypeptides further comprisinganionic or neutral amino acids, such as histone, collagen; and syntheticor natural peptides.

In one embodiment, the cationic polymer comprises a polypeptide having achemical modification at a terminal residue. In one embodiment, thecationic polymer comprises a polypeptide having a modification theN-terminus. In one embodiment, the cationic polymer comprises apolypeptide having a modification at the C-terminus. In one embodiment,the cationic polymer comprises a polypeptide having a free carboxylate(—O⁻) or carboxylic acid (—OH) at the C-terminus. In one embodiment, thecationic polymer comprises a polypeptide having an amido group (—NH₂) atthe C-terminus. In one embodiment, the cationic polymer comprises apolypeptide comprising a sequence selected from the group consisting ofFFIYVWRRR (SEQ ID NO:1), FIKWKFRWWKWRK (SEQ ID NO:2), HQFRFRFRVRRK (SEQID NO:3), ILPWKWRWWKWRR (SEQ ID NO:4), ILRWKWRWWRWRR (SEQ ID NO:5),IRMRIRVLL (SEQ ID NO:6), KFKWWRMLI (SEQ ID NO:7), KIWWWWRKR (SEQ IDNO:8), KRKKRFKWW (SEQ ID NO:9), KRRWRIWLV (SEQ ID NO:10), KRWHWWRRHWVVW(SEQ ID NO:11), KRWWKWWRR (SEQ ID NO:12), KRWWRKWWR (SEQ ID NO:13),KRWWWWRFR (SEQ ID NO:14), KWKWWWRKI (SEQ ID NO:15), LKRRWKWWI (SEQ IDNO:16), LRFILWWKR (SEQ ID NO:17), LRRWIRIRW (SEQ ID NO:18), NWRKLYRRK(SEQ ID NO:19), RIKRWWWWR (SEQ ID NO:20), RIRRWKFRW (SEQ ID NO:21),RKFRWWVIR (SEQ ID NO:22), RKWKIKWYW (SEQ ID NO:23), RLKRWWKFL (SEQ IDNO:24), RLRRIVVIRVFR (SEQ ID NO:25), RLWRIVVIRVKR (SEQ ID NO:26),RLWWKIWLK (SEQ ID NO:27), RLWWWWRRK (SEQ ID NO:28), RQRRVVIWW (SEQ IDNO:29), RRRIKIRWY (SEQ ID NO:30), RRRWWKLMM (SEQ ID NO:31), RRWKIVVIRWRR(SEQ ID NO:32), RRWRVIVKW (SEQ ID NO:33), RRWWKWWWR (SEQ ID NO:34),RRWWRWVVW (SEQ ID NO:35), RRYHWRIYI (SEQ ID NO:36), RTKKWIVWI (SEQ IDNO:37), RWRRKWWWW (SEQ ID NO:38), RWRWWWRVY (SEQ ID NO:39), RWWIRIRWH(SEQ ID NO:40), RWWRKIWKW (SEQ ID NO:41), RWWRWRKWW (SEQ ID NO:42),VRLRIRVRVIRK (SEQ ID NO:43), WFKMRWWGR (SEQ ID NO:44), WKIVFWWRR (SEQ IDNO:45), WKWLKKWIK (SEQ ID NO:46), WKWRVRVTI (SEQ ID NO:47), WRKFWKYLK(SEQ ID NO:48), YKFRWRIYI (SEQ ID NO:49), YRLRVKWKW (SEQ ID NO:50), andcombinations and/or C-terminal amido variants thereof.

Exemplary C-terminal amido-modified peptides include, but are notlimited to, ILRWKWRWWRWRR-NH₂ (SEQ ID NO:51), KRWHWWRRHWVVW-NH₂ (SEQ IDNO: 52), KRWWKWWRR-NH₂ (SEQ ID NO:53), RRWWRWVVW-NH₂ (SEQ ID NO:54), andWKWLKKWIK-NH₂ (SEQ ID NO:55).

In some embodiments, the cationic polypeptide is an antimicrobialpeptide. In some embodiments, the cationic peptide is anantimycobacterial peptide. In one embodiment, the cationic polypeptidecomprises the sequence ILRWKWRWWRWRR (SEQ ID NO:5), KRWHWWRRHWVVW (SEQID NO:11), KRWWKWWRR (SEQ ID NO:12), RRWWRWVVW (SEQ ID NO:35), orWKWLKKWIK (SEQ ID NO:46).

In one embodiment, the cationic polymer comprises a polypeptide having alabel at the C-terminus. In one embodiment, the label is selected fromthe group consisting of an affinity label molecule, a photoaffinitylabel, a dye, a chromophore, a fluorescent molecule, a phosphorescentmolecule, a chemiluminescent molecule, an energy transfer agent, aphotocrosslinker molecule, a redox-active molecule, an isotopic labelmolecule, a spin label molecule, a metal chelator, a metal-comprisingmoiety, a heavy atom-comprising-moiety, a radioactive moiety, a contrastagent molecule, a MRI contrast agent, an isotopically labeled molecule,a PET agent, a polypeptide, a cell penetrating polypeptide, acarbohydrate, a polynucleotide, a peptide nucleic acid, a fatty acid, alipid, biotin, a biotin analogue, a polymer, a small molecule, a drug ordrug candidate, a cytotoxic molecule, a solid support, a surface, aresin, a nanoparticle, a quantum dot and any combination thereof.

The size/length of the cationic polymer is not particularly limited. Inone embodiment, the cationic polymer comprises about 10 monomericrepeating units. In one embodiment, the cationic polymer comprises about20 monomeric repeating units. In one embodiment, the cationic polymercomprises about 30 monomeric repeating units. In one embodiment, thecationic polymer comprises about 50 monomeric repeating units. In oneembodiment, the cationic polymer comprises about 100 monomeric repeatingunits. In one embodiment, the cationic polymer comprises about 250monomeric repeating units. In one embodiment, the cationic polymercomprises about 400 monomeric repeating units. In one embodiment, thecationic polymer comprises about 800 monomeric repeating units.

In some embodiments, the cationic polymer is a cationic peptide. Thepeptide of the present invention may be made using chemical methods. Forexample, peptides can be synthesized by solid phase techniques (RobergeJ Y et al (1995) Science 269: 202-204), cleaved from the resin, andpurified by preparative high performance liquid chromatography.Automated synthesis may be achieved, for example, using the ABI 431 APeptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer. Representative methods for preparing thepeptides of the invention are provided in Example 4-6.

The invention should also be construed to include any form of a peptidehaving substantial homology to a peptide disclosed herein. Preferably, apeptide which is “substantially homologous” is about 50% homologous,more preferably about 70% homologous, even more preferably about 80%homologous, more preferably about 90% homologous, even more preferably,about 95% homologous, and even more preferably about 99% homologous toamino acid sequence of a peptide disclosed herein.

The peptide may alternatively be made by recombinant means or bycleavage from a longer polypeptide. The composition of a peptide may beconfirmed by amino acid analysis or sequencing.

The variants of the peptides according to the present invention may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, (ii) one in whichthere are one or more modified amino acid residues, e.g., residues thatare modified by the attachment of substituent groups, (iii) one in whichthe peptide is an alternative splice variant of the peptide of thepresent invention, (iv) fragments of the peptides and/or (v) one inwhich the peptide is fused with another peptide, such as a leader orsecretory sequence or a sequence which is employed for purification (forexample, His-tag) or for detection (for example, Sv5 epitope tag). Thefragments include peptides generated via proteolytic cleavage (includingmulti-site proteolysis) of an original sequence. Variants may bepost-translationally, or chemically modified. Such variants are deemedto be within the scope of those skilled in the art from the teachingherein.

As known in the art the “similarity” between two peptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to a sequence of a second polypeptide.Variants are defined to include peptide sequences different from theoriginal sequence, preferably different from the original sequence inless than 40% of residues per segment of interest, more preferablydifferent from the original sequence in less than 25% of residues persegment of interest, more preferably different by less than 10% ofresidues per segment of interest, most preferably different from theoriginal protein sequence in just a few residues per segment of interestand at the same time sufficiently homologous to the original sequence topreserve the functionality of the original sequence. The presentinvention includes amino acid sequences that are at least 60%, 65%, 70%,72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to theoriginal amino acid sequence. The degree of identity between twopeptides is determined using computer algorithms and methods that arewidely known for the persons skilled in the art. The identity betweentwo amino acid sequences is preferably determined by using the BLASTPalgorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda,Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

The peptides of the invention can be post-translationally modified. Forexample, post-translational modifications that fall within the scope ofthe present invention include signal peptide cleavage, glycosylation,acetylation, isoprenylation, proteolysis, myristoylation, proteinfolding and proteolytic processing, etc. Some modifications orprocessing events require introduction of additional biologicalmachinery. For example, processing events, such as signal peptidecleavage and core glycosylation, are examined by adding caninemicrosomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489)to a standard translation reaction.

The peptides of the invention may include unnatural amino acids formedby post-translational modification or by introducing unnatural aminoacids during translation. A variety of approaches are available forintroducing unnatural amino acids during protein translation.

A peptide or protein of the invention may be conjugated with othermolecules, such as proteins, to prepare fusion proteins. This may beaccomplished, for example, by the synthesis of N-terminal or C-terminalfusion proteins provided that the resulting fusion protein retains thefunctionality of a peptide of the invention.

A peptide or protein of the invention may be phosphorylated usingconventional methods such as the method described in Reedijk et al. (TheEMBO Journal 11(4):1365, 1992).

Cyclic derivatives of the peptides of the invention are also part of thepresent invention. Cyclization may allow the peptide to assume a morefavorable conformation for association with other molecules. Forexample, cyclization may allow the peptide to assume a more favorableconformation for association with a target protein. Accordingly,cyclization may result in improved binding affinity and specificitytoward the target protein. Cyclization may also confer to the peptidebeneficial properties such as increased resistance against proteolysis,increased cell permeability, and/or more favorable pharmacokineticproperties such as oral bioavailability, reduced clearance, and thelike. In one embodiment, the cyclization of a peptide of the inventionstabilizes the peptide into an α-helical conformation.

Cyclization may be achieved using techniques known in the art. Thesemethods include the use of covalent inter-side-chain linkages such asdisulfide bonds (Jackson, King et al. 1991), lactam (Osapay and Taylor1992), thioether (Brunel and Dawson 2005) or triazole (Scrima, LeChevalier-Isaad et al. 2010; Kawamoto, Coleska et al. 2012) bridges,‘hydrocarbon staples’ (Blackwell and Grubbs 1998; Schafmeister, Po etal. 2000; Bernal, Wade et al. 2010), and cysteine cross-linking moieties(Zhang, Sadovski et al. 2007; Muppidi, Wang et al. 2011; Jo, Meinhardtet al. 2012; Spokoyny, Zou et al. 2013). Another known approach forstabilization of α-helical peptides involves the introduction ofso-called ‘hydrogen bond surrogates’, i.e. hydrocarbon linkagesreplacing an N-terminal i/i+4 hydrogen bond (Wang, Liao et al. 2005).Any of these methods, or combination thereof, can be applied tostabilize the a-helical conformation of PGC1β, PGC1α-, or PRC-derivedpeptides for the purpose of developing inhibitors of the interactionbetween CBP80 and members of the PGC1 family of co-activators.

Other methods of cyclization disulfide bonds which may be formed betweentwo appropriately spaced components having free sulfhydryl groups, or anamide bond may be formed between an amino group of one component and acarboxyl group of another component. Cyclization may also be achievedusing an azobenzene-containing amino acid as described by Ulysse, L., etal., J. Am. Chem. Soc. 1995, 117, 8466-8467. The components that formthe bonds may be side chains of amino acids, non-amino acid componentsor a combination of the two. In an embodiment of the invention, cyclicpeptides may comprise a beta-turn in the right position. Beta-turns maybe introduced into the peptides of the invention by adding the aminoacids Pro-Gly at the right position.

It may be desirable to produce a cyclic peptide which is more flexiblethan the cyclic peptides containing peptide bond linkages as describedabove. A more flexible peptide may be prepared by introducing cysteinesat the right and left position of the peptide and forming a disulphidebridge between the two cysteines. The two cysteines are arranged so asnot to deform the beta-sheet and turn. The peptide is more flexible as aresult of the length of the disulfide linkage and the smaller number ofhydrogen bonds in the beta-sheet portion. The relative flexibility of acyclic peptide can be determined by molecular dynamics simulations.

The invention also relates to peptides comprising a peptide fused to, orintegrated into, a target protein, and/or a targeting domain capable ofdirecting the chimeric protein to a desired cellular component or celltype or tissue. The chimeric proteins may also contain additional aminoacid sequences or domains. The chimeric proteins are recombinant in thesense that the various components are from different sources, and assuch are not found together in nature (i.e., are heterologous).

In one embodiment, the targeting domain can be a membrane spanningdomain, a membrane binding domain, or a sequence directing the proteinto associate with for example vesicles or with the nucleus. In oneembodiment, the targeting domain can target a peptide to a particularcell type or tissue. For example, the targeting domain can be a cellsurface ligand or an antibody against cell surface antigens of a targettissue (e.g., bone, regenerating bone, degenerating bone, cartilage). Atargeting domain may target the peptide of the invention to a cellularcomponent.

A peptide of the invention may be synthesized by conventionaltechniques. For example, the peptides or chimeric proteins may besynthesized by chemical synthesis using solid phase peptide synthesis.These methods employ either solid or solution phase synthesis methods(see for example, J. M. Stewart, and J. D. Young, Solid Phase PeptideSynthesis, 2^(nd) Ed., Pierce Chemical Co., Rockford Ill. (1984) and G.Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biologyeditors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York,1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky,Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E.Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, suprs, Vol 1, for classical solution synthesis). By way ofexample, a peptide of the invention may be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry with direct incorporationof phosphothreonine as theN-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

N-terminal or C-terminal fusion proteins comprising a peptide orchimeric protein of the invention conjugated with other molecules may beprepared by fusing, through recombinant techniques, the N-terminal orC-terminal of the peptide or chimeric protein, and the sequence of aselected protein or selectable marker with a desired biologicalfunction. The resultant fusion proteins contain the peptide fused to theselected protein or marker protein as described herein. Examples ofproteins which may be used to prepare fusion proteins includeimmunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

Peptides of the invention may be developed using a biological expressionsystem. The use of these systems allows the production of largelibraries of random peptide sequences and the screening of theselibraries for peptide sequences that bind to particular proteins.Libraries may be produced by cloning synthetic DNA that encodes randompeptide sequences into appropriate expression vectors (see Christian etal 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404;Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries mayalso be constructed by concurrent synthesis of overlapping peptides (seeU.S. Pat. No. 4,708,871).

The peptides and chimeric proteins of the invention may be convertedinto pharmaceutical salts by reacting with inorganic acids such ashydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid,etc., or organic acids such as formic acid, acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid,malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid,benezenesulfonic acid, and toluenesulfonic acids.

In one embodiment, at least one of the anionic polymer and the cationicpolymer is registered as a GRAS (generally recognized as safe)substance. In one embodiment, at least one of the anionic polymer andthe cationic polymer is degraded by bacterial enzymes. In oneembodiment, at least one of the anionic polymer and the cationic polymeris degraded by enzymes produced by bacteria but not produced by humans.

In some embodiments, the particles of the present invention arecore-shell particles. In one embodiment, the core comprises the cationicpolymer. In another embodiment, the core comprises the anionic polymer.In one embodiment, the shell comprises the anionic polymer. In anotherembodiment, the shell comprises the cationic polymer. In one embodiment,the cationic polymer coats the surface of the particles.

In some embodiments, the anionic polymer and the cationic polymer arebound via electrostatic interactions. In one embodiment, the cationicpolymer is a crosslinking agent in the anionic polymer matrix. In oneembodiment, the anionic polymer and the cationic polymer are notcovalently bound. In another embodiment, the anionic polymer and thecationic polymer are covalently bound. In one embodiment, the cationicpolymer is covalently bound to the surface the drug delivery particles.

In one embodiment, the ratio of negative charge in the anionic polymer(N) to positive charge in the cationic polymer (P), N:P, is betweenabout 1:1 and about 15:1. In one embodiment, the ratio N:P is about 1:1.In one embodiment, the ratio N:P is about 2.5:1. In one embodiment, theratio N:P is about 5:1. In one embodiment, the ratio N:P is about 7.5:1.In one embodiment, the ratio N:P is about 10:1. In one embodiment, theratio N:P is about 12.5:1. In one embodiment, the ratio N:P is about15:1.

In some embodiments, the size of the particles may change upon exposureto water or to physiologic media. In some embodiments, the particlesexpand to up to about 3 times their original size in water orphysiologic media. In some embodiments, the particles expand up to about2 times their original size in water or physiologic media. In oneembodiment, the size of the swollen particles depends on the N:P ratioof the particles. In some embodiments, the particles of the presentinvention degrade after a certain time period in physiologic media. Inone embodiment, the particles degrade after between 0.5 and 80 hours. Inone embodiment, the particles degrade after about 0.5 hours. In oneembodiment, the particles degrade after about 4 hours. In oneembodiment, the particles degrade after about 20 hours. In oneembodiment, the particles degrade after about 50 hours. In oneembodiment, the particles degrade after about 70 hours. In oneembodiment, the particles degrade after about 80 hours. In oneembodiment, the time that it takes the particles to degrade depends onthe N:P ratio of the particles.

The biologically active agent can be any biologically active agent thatwould be appreciated by one of skill in the art. Exemplary bioactivecompounds include, but are not limited to, antimicrobial compounds,anti-cancer compounds, antiviral compounds, and monoclonal antibodies orother biologics.

Non-limiting examples of antimicrobial compounds are levofloxacin,aminosalicycic acid, capreomycin, ethambutol, isoniazid, pyrazinamide,rifabutin, rifampin, clofazime, doxycycline, neomycin, clindamycin,minocycline, gentamycin, rifampicin, chlorhexidine, chloroxylenol,methylisothizolone, thymol, a-terpineol, cetylpyridinium chloride,hexachlorophene, triclosan, nitrofurantoin, erythromycin, nafcillin,cefazolin, imipenem, astreonam, gentamicin, sulfamethoxazole,vancomycin, ciprofloxacin, trimethoprim, rifampin, metronidazole,clindamycin, teicoplanin, mupirocin, azithromycin, clarithromycin,ofoxacin, lomefloxacin, norfloxacin, nalidixic acid, sparfloxacin,pefloxacin, amifloxacin, gatifloxacin, moxifloxacin, gemifloxacin,enoxacin, fleroxacin, minocycline, linexolid, temafloxacin,tosufloxacin, clinafloxacin, sulbactam, clavulanic acid, amphotericin B,fluconazole, itraconazole, ketoconazole, nystatin, penicillins,cephalosporins, carbepenems, beta-lactams antibiotics, aminoglycosides,macrolides, lincosamides, glycopeptides, tetracylines, chloramphenicol,quinolones, fucidines, sulfonamides, trimethoprims, rifamycins,oxalines, streptogramins, lipopeptides, ketolides, polyenes, azoles,echinocandines, and any combination(s) thereof. In one embodiment, theantimicrobial compound is an antimycobacterial compound. In oneembodiment, the composition comprises a multidrug cocktail. In oneembodiment, the multidrug cocktail comprises rifampicin, isoniazid,pyrazinamide and ethambutol.

In one embodiment, the antimicrobial compound is an inhibitor oftrans-translation or protein synthesis. Exemplary inhibitors include,but are not limited to, kanamycin, gentamicin, hygromycin B,streptomycin, G418, paromomycin, spectinomycin, fusidic acid,thiostrepton, GE2270A, GE37468, erythromycin, spiramycin, tylosin,KKL-10, KKL-22, KKL-35, KKL-52, and KKL-55.

Non-limiting examples of anti-cancer compounds are acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin;

ametantrone acetate; aminoglutethimide; amsacrine; anastrozole;anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin;batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafidedimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine;busulfan; cactinomycin; calusterone; caracemide; carbetimer;carboplatin; carmustine; carubicin hydrochloride; carzelesin;cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatolmesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine;dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicinhydrochloride; droloxifene; droloxifene citrate; dromostanolonepropionate; duazomycin; edatrexate; eflornithine hydrochloride;elsamitrucin; enloplatin; enpromate; epipropidine; epirubicinhydrochloride; erbulozole; esorubicin hydrochloride; estramustine;estramustine phosphate sodium; etanidazole; etoposide; etoposidephosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide;floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine;fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine;interleukin II (including recombinant interleukin II, or rIL2),interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferonalfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide;

safingol; safingol hydrochloride; semustine; simtrazene; sparfosatesodium; sparsomycin; spirogermanium hydrochloride; spiromustine;spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride. Other anti-cancer drugs include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA;

bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dihydrotaxol; dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. In one embodiment, the anti-cancer drug is doxorubicin.

Exemplary antiviral drugs include, but are not limited to, abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen,arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir,darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz,emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir,fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir,ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine,interferon type iii, interferon type ii, interferon type i, interferon,lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone,nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir(Tamiflu), peginterferon alfa-2a, penciclovir, peramivir, pleconaril,podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir,pyramidine, saquinavir, stavudine, tea tree oil, tenofovir, tenofovirdisoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada,valaciclovir (Valtrex), valganciclovir, vicriviroc, vidarabine,viramidine, zalcitabine, zanamivir (Relenza), and zidovudine.

Exemplary monoclonal antibodies or other biologics include, but are notlimited to 3F8, 8H9, Abagovomab, Abciximab, Adalimumab, Adecatumumab,Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Altumomabpentetate, Amatuximab, Anatumomab mafenatox, Anrukinzumab, Apolizumab,Arcitumomab, Aselizumab, Atinumab, Atlizumab, Atorolimumab,Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab,Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Biciromab,Bivatuzumab mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin,Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumabravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab,CC49, Cedelizumab, Certolizumab pegol, Cetuximab, Citatuzumab bogatox,Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan,Conatumumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab,Daratumumab, Denosumab, Detumomab, Dorlimomab aritox, Drozitumab,Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab,Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab,Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab,Etaracizumab, Etrolizumab, Exbivirumab, Fanolesomab, Faralimomab,Farletuzumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab,Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab,Fresolimumab, Fulranumab, Galiximab, Ganitumab, Gantenerumab,Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab,Glembatumumab vedotin, Golimumab, Gomiliximab, GS6624 , Ibalizumab,Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Inclacumab,Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab,Labetuzumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab,Libivirumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab,Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab,Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab,Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox,Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox,Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab,Nimotuzumab, Nivolumab, Nofetumomab merpentan, Ocrelizumab, Odulimomab,Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumabmonatox, Oregovomab, Otelixizumab, Oxelumab, Ozoralizumab, Pagibaximab,Palivizumab, Panitumumab, Panobacumab, Pascolizumab, Pateclizumab,Patritumab, Pemtumomab, Pertuzumab, Pexelizumab, Pintumomab, Placulumab,Ponezumab, Priliximab, Pritumumab, PRO 140, Quilizumab, Racotumomab,Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab,Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab,Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab,Sarilumab, Satumomab pendetide, Secukinumab, Sevirumab, Sibrotuzumab,Sifalimumab, Siltuximab, Siplizumab, Sirukumab, Solanezumab, Solitomab,Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab,Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomabpaptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab,Teplizumab, Teprotumumab, TGN1412, Ticilimumab, Tigatuzumab, TNX-650,Tocilizumab, Toralizumab, Tositumomab, Tralokinumab, Trastuzumab,TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab,Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab,Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab,Visilizumab, Volociximab, Votumumab, Zalutumumab, Zanolimumab,Ziralimumab, and Zolimomab aritox.

The particles of the present invention may further comprise at least oneexcipient. In one embodiment, the excipient is a pharmaceuticallyacceptable carrier. As used herein, the term “pharmaceuticallyacceptable carrier” means a pharmaceutically acceptable material,composition or carrier, such as a liquid or solid filler, stabilizer,dispersing agent, suspending agent, diluent, excipient, thickeningagent, solvent or encapsulating material, involved in carrying ortransporting a compound useful within the invention within or to thepatient such that it may perform its intended function. Typically, suchconstructs are carried or transported from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation, including the compound useful within the invention,and not injurious to the patient. Some examples of materials that mayserve as pharmaceutically acceptable carriers include: sugars, such aslactose, glucose and sucrose; starches, such as corn starch and potatostarch; cellulose, and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients, such as cocoa butter and suppositorywaxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; surfaceactive agents; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; phosphate buffer solutions; and othernon-toxic compatible substances employed in pharmaceutical formulations.As used herein, “pharmaceutically acceptable carrier” also includes anyand all coatings, antibacterial and antifungal agents, and absorptiondelaying agents, and the like that are compatible with the activity ofthe compound useful within the invention, and are physiologicallyacceptable to the patient. Supplementary active compounds may also beincorporated into the compositions. The “pharmaceutically acceptablecarrier” may further include a pharmaceutically acceptable salt of thecompound useful within the invention. Other additional ingredients thatmay be included in the pharmaceutical compositions used in the practiceof the invention are known in the art and described, for example inRemington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co.,1985, Easton, Pa.), which is incorporated herein by reference.

In one embodiment, the biologically active agent is localized, oressentially localized, in the core of the core-shell particles. In oneembodiment, the biologically active agent is localized, or essentiallylocalized, in the shell of the core-shell particles. In one embodiment,the biologically active agent is localized, or essentially localized, inthe core and the shell of the core-shell particles. In one embodiment,the biologically active agent is localized, or essentially localized, onthe outer surface of the core-shell particles.

In one embodiment, the biologically active agent is not covalently boundto the cationic polymer or to the anionic polymer. In one embodiment,the biologically active agent is covalently bound to the cationicpolymer via a tether. In one embodiment, the biologically active agentis covalently bound to the anionic polymer via a tether. In oneembodiment, the biologically active agent is covalently bound to boththe cationic polymer and the anionic polymer via at least tether. In oneembodiment, the tether degrades under physiologic conditions. In oneembodiment, the tether comprises at least one of a polypeptide, apolysaccharide, a poly lactic acid, or a polyanhydride.

In one embodiment, the biologically active agent is released underphysiological conditions, such as in physiologic media. In oneembodiment, the biologically active agent is released at a raterepresented by zero-order kinetics. In one embodiment, the biologicallyactive agent is released at a rate represented by first-order kinetics.In one embodiment, the biologically active is retained until theparticles degrade. In one embodiment, the release rate of thebiologically active agent can be tuned through localization of the agentwithin the particles, the N:P ratio of the particles, and thepresence/absence of additional cationic peptides.

In one embodiment, the particles of the present invention are generallyspherical in shape. In one embodiment, the particles of the presentinvention have a ruffled surface. In another embodiment, the particleshave a smooth surface. There is no particular limit to the size of theparticles of the present invention. In some embodiments, the particleshave diameter between 50 and 5000 nm. In some embodiments, the particleshave a diameter between 50 and 200 nm. In one embodiment, the particleshave a diameter of about 50 nm. In one embodiment, the particles have adiameter of about 60 nm. In one embodiment, the particles have adiameter of about 80 nm. In one embodiment, the particles have adiameter of about 100 nm. In one embodiment, the particles have adiameter of about 120 nm. In one embodiment, the particles have adiameter of about 140 nm. In one embodiment, the particles have adiameter of about 160 nm. In one embodiment, the particles have adiameter of about 180 nm. In one embodiment, the particles have adiameter of about 200 nm. In some embodiments, the particles have adiameter between 500 and 5000 nm. In one embodiment, the particles havea diameter between 1000 and 3000 nm. In one embodiment, the particleshave a diameter of about 200 nm. In one embodiment, the particles have adiameter of about 500 nm. In one embodiment, the particles have adiameter of about 750 nm. In one embodiment, the particles have adiameter of about 1000 nm. In one embodiment, the particles have adiameter of about 1250 nm. In one embodiment, the particles have adiameter of about 1500 nm. In one embodiment, the particles have adiameter of about 1750 nm. In one embodiment, the particles have adiameter of about 2000 nm. In one embodiment, the particles have adiameter of about 2250 nm. In one embodiment, the particles have adiameter of about 2500 nm. In one embodiment, the particles have adiameter of about 2750 nm. In one embodiment, the particles have adiameter of about 3000 nm.

In some embodiments, the outer surface of the particles exhibits anegative charge. In other embodiments, the outer surface of theparticles exhibits a positive charge. In other embodiments, the outersurface of the particles is neutral. In one embodiment, the charge ofthe outer surface of the particles can be measured by its zetapotential. In one embodiment, the zeta potential of the particles isbetween −50 and 50 mV. In one embodiment, the zeta potential of theparticles is between −50 and 30 mV. In one embodiment, the zetapotential of the particles is about −50 mV. In one embodiment, the zetapotential of the particles is about −40 mV. In one embodiment, the zetapotential of the particles is about −30 mV. In one embodiment, the zetapotential of the particles is about −20 mV. In one embodiment, the zetapotential of the particles is about −10 mV. In one embodiment, the zetapotential of the particles is about 0 mV. In one embodiment, the zetapotential of the particles is about 10 mV. In one embodiment, the zetapotential of the particles is about 20 mV. In one embodiment, the zetapotential of the particles is about 30 mV.

Methods of Making

In one aspect, the present invention relates to a method of forming aplurality of drug delivery particles. An exemplary method is provided inFIG. 100. In step 110, a sample solution comprising at least one anionicpolymer is provided. In step 120, a bath solution comprising at leastone cationic polymer is provided. In step 130, the sample solution iselectrosprayed into the bath solution to form a drug delivery particlesolution. In step 140, the drug delivery particles are isolated from thedrug delivery particle solution. At least one of the sample solution,the bath solution, or the solution of drug delivery particles furthercomprises at least one biologically active agent.

Electrospraying is a technique in which a voltage is applied to thesample solution as it is passed through a capillary tip; coulombicrepulsion within the ejected solution generates a fine mist that iscollected in the bath solution. In some embodiments, the voltage appliedto the sample solution is between 1 and 100 kV. In one embodiment, thevoltage applied to the sample solution is between 10 and 50 kV. In oneembodiment, the voltage applied to the sample solution is about 10 kV.In one embodiment, the voltage applied to the sample solution is about15 kV. In one embodiment, the voltage applied to the sample solution isabout 20 kV. In one embodiment, the voltage applied to the samplesolution is about 25 kV. In one embodiment, the voltage applied to thesample solution is about 30 kV. In one embodiment, the voltage appliedto the sample solution is about 35 kV. In one embodiment, the voltageapplied to the sample solution is about 40 kV. In one embodiment, thevoltage applied to the sample solution is about 45 kV. In oneembodiment, the voltage applied to the sample solution is about 50 kV.

In one embodiment, the spray rate of the sample solution into the bathsolution is between about 0.05 mL/min and about 1 mL/min. In oneembodiment, the spray rate of the sample solution into the bath solutionis about 0.1 mL/min.

The sample solution can comprise any anionic polymer, including, but notlimited, to, anionic polymers disclosed herein. In one embodiment, thesample solution comprises between about 0.1 wt % anionic polymer andabout 4 wt % anionic polymer. In one embodiment, the sample solutioncomprises about 0.1 wt % anionic polymer. In one embodiment, the samplesolution comprises about 0.2 wt % anionic polymer. In one embodiment,the sample solution comprises about 0.3 wt % anionic polymer. In oneembodiment, the sample solution comprises about 0.4 wt % anionicpolymer. In one embodiment, the sample solution comprises about 0.5 wt %anionic polymer. In one embodiment, the sample solution comprises about0.6 wt % anionic polymer. In one embodiment, the sample solutioncomprises about 0.7 wt % anionic polymer. In one embodiment, the samplesolution comprises about 0.8 wt % anionic polymer. In one embodiment,the sample solution comprises about 0.9 wt % anionic polymer. In oneembodiment, the sample solution comprises about 1.0 wt % anionicpolymer. In one embodiment, the sample solution comprises about 1.5 wt %anionic polymer. In one embodiment, the sample solution comprises about2.0 wt % anionic polymer. In one embodiment, the sample solutioncomprises about 2.5 wt % anionic polymer. In one embodiment, the samplesolution comprises about 3.0 wt % anionic polymer. In one embodiment,the sample solution comprises about 3.5 wt % anionic polymer. In oneembodiment, the sample solution comprises about 4.0 wt % anionicpolymer.

The bath solution can comprise any cationic polymer, including, but notlimited to, cationic polymers disclosed herein. In one embodiment, theconcentration of the bath solution comprising the cationic polymer isbetween 0.001% and 0.1% w/v. In one embodiment, the concentration to thebath solution is between 0.01% and 0.1% w/v. In one embodiment, theconcentration of the bath solution is about 0.01% w/v. In oneembodiment, the concentration of the bath solution is between 0.5 and 3mg/mL. In one embodiment, the concentration of the bath solution isabout 1 mg/mL.

In one embodiment, any of the bath solution, sample solution, or drugdelivery particle solution further comprises up to about 5% v/v DMSO. Inone embodiment, any of the bath solution, sample solution, or drugdelivery particle solution further comprises a pharmaceuticallyacceptable carrier as described elsewhere herein.

In one embodiment, the step of electrospraying the sample solution intothe bath solution to form a drug delivery particle solution furthercomprises the step of incubating the drug delivery solution for at leastone hour at least 37° C.

In one embodiment, the step of isolating a plurality of drug deliveryparticles from the drug delivery particle solution comprises the step ofcentrifuging the drug delivery particle solution and removing thesupernatant. In one embodiment, centrifugation may generate particles ofsize between about 50 nm and about 300 nm. In one embodiment, the stepof isolating a plurality of drug delivery particles from the drugdelivery particle solution comprises the step of dialyzing the particlesfrom the drug delivery particle solution. In one embodiment, dialysismay generate larger particles than centrifugation. In one embodiment,dialysis may generate particles of size between about 300 nm and about1000 nm. In one embodiment, the step of isolating a plurality of drugdelivery particles from the drug delivery particle solution comprisesthe step of filtering the drug delivery particle solution. In oneembodiment, the step of isolating a plurality of drug delivery particlesfrom the drug delivery particle solution further comprises the step oflyophilizing the drug delivery particles to form a dry powder.

Methods of Treatment

In one aspect, the present invention relates to a method of treating amycobacterial infection in a subject in need thereof, the methodcomprising the step of administering to the subject a formulationcomprising the drug delivery particles of the present invention. In oneembodiment, administration of a formulation comprising the drug deliveryparticles of the present invention prior to exposure to mycobacteria canprevent infection.

In one embodiment, the particles of the present invention are preparedas dry powder formulation. In one embodiment, dry powder is suitable forinhalation formulation. In one embodiment, the dry powder formulationcomprises particles with a mass median aerodynamic diameter (MMAD)between about 1 and about 5 μm. In one embodiment, the dry powderformulation comprises particles with an MMAD of about 1.5 μm. In oneembodiment, the dry powder formulation comprises particles having anegative surface charge, as discussed elsewhere herein.

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions comprising drugdelivery particles of the present invention. The pharmaceuticalcompositions may be suitable for a variety of modes of administrationdescribed herein, including for example systemic or localizedadministration. The pharmaceutical compositions can be in the form ofeye drops, injectable solutions, or in a form suitable for inhalation(either through the mouth or the nose) or oral administration. Thepharmaceutical compositions described herein can be packaged in singleunit dosages or in multidosage forms.

In some embodiments, the pharmaceutical compositions comprise apharmaceutically acceptable carrier suitable for administration tohuman. In some embodiments, the pharmaceutical compositions comprise apharmaceutically acceptable carrier suitable for intraocular injection.In some embodiments, the pharmaceutical compositions comprise apharmaceutically acceptable carrier suitable for topical application. Insome embodiments, the pharmaceutical compositions comprise apharmaceutically acceptable carrier suitable for intravenous injection.In some embodiments, the pharmaceutical compositions comprise and apharmaceutically acceptable carrier suitable for injection into thearteries.

The compositions are generally formulated as sterile, substantiallyisotonic, and in full compliance with all Good Manufacturing Practice(GMP) regulations of the U.S. Food and Drug Administration. In someembodiments, the composition is free of pathogen. For injection, thepharmaceutical composition can be in the form of liquid solutions, forexample in physiologically compatible buffers such as Hank's solution orRinger's solution. In addition, the drug delivery particlepharmaceutical composition can be in a solid form and redissolved orsuspended immediately prior to use. Lyophilized compositions are alsoincluded.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate).Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

The present invention in some embodiments provides compositionscomprising drug delivery particles and a pharmaceutically acceptablecarrier suitable for administration to the eye. Such pharmaceuticalcarriers can be sterile liquids, such as water and oil, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, and the like. Saline solutions and aqueousdextrose, polyethylene glycol (PEG) and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, sodium state, glycerol monostearate,glycerol, propylene, water, and the like. The pharmaceuticalcomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. The drug delivery particlesand other components of the composition may be encased in polymers orfibrin glues to provide controlled release of the molecule. Thesecompositions can take the form of solutions, suspensions, emulsions,ointment, gel, or other solid or semisolid compositions, and the like.The compositions typically have a pH in the range of 4.5 to 8.0. Thecompositions must also be formulated to have osmotic values that arecompatible with the aqueous humor of the eye and ophthalmic tissues.Such osmotic values will generally be in the range of from about 200 toabout 400 milliosmoles per kilogram of water (“mOsm/kg”), but willpreferably be about 300 mOsm/kg.

In some embodiments, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted for injectionintravenously, intraperitoneally, or intravitreally. Typically,compositions for injection are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions may further comprise additional ingredients, forexample preservatives, buffers, tonicity agents, antioxidants andstabilizers, nonionic wetting or clarifying agents, viscosity-increasingagents, and the like.

Suitable preservatives for use in a solution include polyquaternium-1,benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propylparaben, phenylethyl alcohol, edetate disodium, sorbic acid,benzethonium chloride, and the like. Typically (but not necessarily),such preservatives are employed at a level of from 0.001% to 1.0% byweight.

Suitable buffers include boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium carbonate, sodiumacetate, sodium biphosphate and the like, in amounts sufficient tomaintain the pH at between about pH 6 and pH 8, and preferably, betweenabout pH 7 and pH 7.5.

Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin,potassium chloride, propylene glycol, sodium chloride, and the like,such that the sodium chloride equivalent of the ophthalmic solution isin the range 0.9 plus or minus 0.2%.

Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, thiourea and the like. Suitablewetting and clarifying agents include polysorbate 80, polysorbate 20,poloxamer 282 and tyloxapol. Suitable viscosity-increasing agentsinclude dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like.

The use of viscosity enhancing agents to provide topical compositionswith viscosities greater than the viscosity of simple aqueous solutionsmay be desirable. Such viscosity building agents include, for example,polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxy propyl cellulose or other agents know to those skilled in theart. Such agents are typically employed at a level of from 0.01% to 2%by weight.

In some embodiments, there is provided a pharmaceutical composition fordelivery of a nucleotide encapsulated in a drug delivery particle. Thepharmaceutical composition for gene therapy can be in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle or compound is imbedded. Alternatively, where thecomplete gene delivery system can be produced intact from recombinantcells, e.g., retroviral vectors, the pharmaceutical composition cancomprise one or more cells which produce the gene delivery system.

In clinical settings, a gene delivery system for a gene therapeutic canbe introduced into a subject by any of a number of methods. Forinstance, a pharmaceutical composition of the gene delivery system canbe introduced systemically, e.g., by intravenous injection, and specifictransduction of the protein in the target cells occurs predominantlyfrom specificity of transfection provided by the gene delivery vehicle,cell-type or tissue-type expression due to the transcriptionalregulatory sequences controlling expression of the receptor gene, or acombination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced by catheter, See U.S. Pat. No. 5,328,470, or by stereotacticinjection, Chen et al. (1994), Proc. Natl. Acad. Sci., USA 91:3054-3057.

Administration

The compositions described herein can be administered to an individualvia any route, including, but not limited to, intravenous (e.g., byinfusion pumps), intraperitoneal, intraocular, intra-arterial,intrapulmonary, oral, intravesicular, intramuscular, intra-tracheal,subcutaneous, intrathecal, transdermal, transpleural, topical,inhalational (e.g., as mists of sprays dry powders, or aerosols),mucosal (such as via nasal mucosa), gastrointestinal, intraarticular,intracisternal, intraventricular, rectal (i.e., via suppository),vaginal (i.e., via pessary), intracranial, intraurethral, intrahepatic,and intratumoral. In some embodiments, the compositions are administeredsystemically (for example by intravenous injection). In someembodiments, the compositions are administered locally (for example byintraarterial or intraocular injection). In some embodiments, thecompositions are administered by ex vivo incubation or perfusion.

Dosing

The optimal effective amount of the compositions can be determinedempirically and will depend on the type and severity of the disease,route of administration, disease progression and health, mass and bodyarea of the individual. Such determinations are within the skill of onein the art. The effective amount can also be determined based on invitro complement activation assays. Examples of dosages of drug deliveryparticles which can be used for methods described herein include, butare not limited to, an effective amount within the dosage range of anyof about 0.01 mg/kg to about 300 mg/kg, or within about 0.1 mg/kg toabout 40 mg/kg, or with about 1 mg/kg to about 20 mg/kg, or within about1 mg/kg to about 10 mg/kg. In some embodiments, the amount ofbiologically active agent administered to an individual is about 10 mgto about 500 mg per dose, including for example any of about 10 mg toabout 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg,about 200 mg to about 300 mg, about 300 mg to about 500 mg, about 500 mgto about 1 mg, about 1 mg to about 10 mg, about 10 mg to about 50 mg,about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mgto about 300 mg, about 300 mg to about 400 mg, or about 400 mg to about500 mg per dose.

The compositions comprising drug delivery particles may be administeredin a single daily dose, or the total daily dose may be administered individed dosages of two, three, or four times daily. The compositions canalso be administered less frequently than daily, for example, six timesa week, five times a week, four times a week, three times a week, twicea week, once a week, once every two weeks, once every three weeks, oncea month, once every two months, once every three months, or once everysix months. The compositions may also be administered in a sustainedrelease formulation, such as in an implant which gradually releases thecomposition for use over a period of time, and which allows for thecomposition to be administered less frequently, such as once a month,once every 2-6 months, once every year, or even a single administration.The drug delivery particles may be administered by injection or surgicalimplantation in various locations.

Dosage amounts and frequency will vary according the particularformulation, the dosage form, and individual patient characteristics.Generally speaking, determining the dosage amount and frequency for aparticular formulation, dosage form, and individual patientcharacteristic can be accomplished using conventional dosing studies,coupled with appropriate diagnostics.

Combination Therapy

In some embodiments, provided pharmaceutical formulations areadministered to a subject in combination with one or more othertherapeutic agents or modalities, for example, useful in the treatmentof one or more diseases, disorders, or conditions treated by therelevant provided pharmaceutical formulation, so the subject issimultaneously exposed to both.

The particular combination of therapies (substances and/or procedures)to employ in a combination regimen will take into account compatibilityof the desired substances and/or procedures and the desired therapeuticeffect to be achieved. In some embodiments, provided compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother therapeutic agents (e.g., desired known antimycobacterialtherapeutics).

It will be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, a therapeutic compound usefulfor mycobacterial infections administered concurrently with acomposition of the present invention), or they may achieve differenteffects (for example, a composition of the present invention may beadministered concurrently with a therapeutic agent that is useful foralleviating adverse side effects, for instance, fever, pain, nausea,etc.). In some embodiments, the composition of the present invention areadministered with a second therapeutic agent.

As used herein, the terms “in combination with” and “in conjunctionwith” mean that the drug delivery particles of the present invention canbe administered concurrently with, prior to, or subsequent to, one ormore other desired therapeutics such as an analgesic, antibacterial,antiviral, anticancer, or biologic agent including but not limited to asub-therapeutic dose of such an agent. In general, each substance willbe administered at a dose and/or on a time schedule determined for thatagent.

In certain embodiments, the method comprises administering a compositioncomprising a combination of an antibacterial agent and the drug deliveryparticles described herein.

In certain embodiments, the method comprises administering one or morecompositions. For example, in one embodiment, the method comprisesadministering a first composition comprising an antibacterial agent anda second composition comprising the drug delivery particles describedherein. The different compositions may be administered to the subject inany order and in any suitable interval. For example, in certainembodiments, the one or more compositions are administeredsimultaneously or near simultaneously. In certain embodiments, themethod comprises a staggered administration of the one or morecompositions, where a first composition is administered and a secondcomposition administered at some later time point. Any suitable intervalof administration which produces the desired therapeutic effect may beused.

In certain embodiments, the method has an additive effect, wherein theoverall effect of the administering a combination of therapeutic agentsor procedures is approximately equal to the sum of the effects ofadministering each therapeutic agent or procedure alone. In otherembodiments, the method has a synergistic effect, wherein the overalleffect of administering a combination of therapeutic agents orprocedures is greater than the sum of the effects of administering eachtherapeutic agent or procedure alone.

Unit Dosages, Articles of Manufacture, and Kits

Also provided are unit dosage forms of drug delivery particlecompositions, each dosage containing from about 0.01 mg to about 50 mg,including for example any of about 0.1 mg to about 50 mg, about 1 mg toabout 50 mg, about 5 mg to about 40 mg, about 10 mg to about 20 mg, orabout 15 mg of the biologically active agent. In some embodiments, theunit dosage forms of drug delivery particles comprise about any of 0.01mg-0.1 mg, 0.1 mg-0.2 mg, 0.2 mg-0.25 mg, 0.25 mg-0.3 mg, 0.3 mg-0.35mg, 0.35 mg-0.4 mg, 0.4 mg-0.5 mg, 0.5 mg-1.0 mg, 10 mg-20 mg, 20 mg-50mg, 50 mg-80 mg, 80 mg-100 mg, 100 mg-150 mg, 150 mg-200 mg, 200 mg-250mg, 250 mg-300 mg, 300 mg-400 mg, or 400 mg-500 mg biologically activeagent. The term “unit dosage form” refers to a physically discrete unitsuitable as unitary dosages for an individual, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical carrier, diluent, or excipient. These unit dosage formscan be stored in suitable packaging in single or multiple unit dosagesand may also be further sterilized and sealed.

The present invention also provides kits comprising compositions (orunit dosages forms and/or articles of manufacture) described herein andmay further comprise instruction(s) on methods of using the composition,such as uses described herein. The kits described herein may furtherinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for performing any methods describedherein.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Example 1: Inhalable Antimicrobials

A new class of inhalable microparticle gels (‘microgels’) formed fromthe electrostatic cross-linking of hyaluronic acid (HA), an FDA approvedanionic polysaccharide (Monheit and Coleman, Dermatol. Ther. 2006, 19(3), 141-150), and cationic antimycobacterial peptides (AMPs) (FIG. 2).The premise of this strategy lies in exploiting the ability of AMPs toelicit rapid and potent lytic destruction of drug-sensitive and-resistant MTb (Ramón-Garcia, et al., Antimicrob. Agents Chemother.2013, 57 (5), 2295-2303), while minimizing their off-target toxicity vialocal aerosol delivery to infected lung tissue. These cationic peptideselicit their activity by preferentially binding to anionic motifs on themicrobe surface and inserting themselves into the bacterial membrane toform a peptide-lipid complex (Gutsmann, Biochim. Biophys. Acta,Biomembr. 2016, 1858 (5), 1034-1043; Zasloff, Nature 2002, 415 (6870),389-395). This perturbation leads to thinning and altered curvature ofthe microbial membrane ultimately resulting in pore formation andbacterial lysis. This mechanism is distinct from conventionalantibiotics that target intracellular pathways involved in bacterialmetabolism and replication, and thus AMPs are effective towardsdrug-resistant MTb. Similarly, the ability of AMPs to disrupt thebacterial cell wall makes them equally as potent towards mycobacteria ina non-proliferative state as they are towards replicating cells.

To compliment this acute activity, degradation of the HA microgel matrixby bacterially secreted enzymes affords long-term release ofencapsulated antibiotics directly to infected lung tissue. In parallel,phagocytic uptake of drug-loaded microgels by lung macrophages allowsfor clearance of pathogens protected within these infected host cellsand associated granulomas. Collectively, this approach affords directdelivery of AMPs and antibiotics to infected lung tissue to elicit rapidand potent killing of MDR- and XDR-TB with minimal side effects, whilecontrollably releasing encapsulated drug to provide durable anti-TBresponses with reduced dosing frequency compared to conventional oralregimens.

Unique to this approach is the ability to synthesize microgels viaelectrostatic cross-linking of AMPs, which are active towardsdrug-resistant bacteria, and biocompatible carbohydrates. Thiseliminates the need for harsh chemicals and cross-linking reagentsduring synthesis, and yields a biomaterial that is inherentlyantimicrobial and likely biocompatible. Further, in addition to theirpotential to kill MDR- and XDR-TB, AMPs have a number of other uniqueproperties rarely enjoyed by traditional small molecule antibiotics.First, it is generally regarded that, due to their rapid andmembrane-specific mechanisms of action, bacterial resistance towardsAMPs is unlikely (Abedinzadeh, et al., J. Antimicrob. Chemother. 2015,70 (5), 1285-1289). Secondly, permeabilization of bacterial membranes byAMPs can enhance the uptake of delivered drugs, thereby increasing theirpotency. Finally, most AMPs are short sequences that, due to advances inmicrowave synthesis (Collins, et al., Organic Letters 2014, 16 (3),940-943), can be prepared quickly, at low cost and in high yield andpurity.

It is worth noting that formulation of AMPs into a particulate carrieris ideally suited for their aerosol delivery to the lung. The cationicnature of AMPs can lead to their adsorption to upper airway mucosalmembranes and thus prevent distribution to infected lower bronchial andalveolar tissue, if not formulated into a micron-sized carrier (Cryan,AAPS J. 2005, 7 (1), E20-E41). Further, encapsulating AMPs within themicrogel matrix serves to inhibit their recognition and destruction byproteases present in the inflammatory diseased lung (Lange, et al., J.Pharm. Sci. 2001, 90 (10), 1647-1657), and minimizes their off-targetdistribution and toxicity towards healthy epithelial tissue and immunecells.

The anionic HA component of microgels also imparts a number of distinctadvantages over traditional particles. Hyaluronidases, the group ofenzymes responsible for HA degradation, are actively secreted by MTb inan effort to utilize HA from the host's extracellular matrix as a carbonsource (Hirayama, et al., PLoS Pathog. 2009, 5 (10), e1000643). Thissuggests that particle degradation and drug release will occurpreferentially to MTb microbes, thereby increasing the effectiveness ofdelivered antibiotics and minimizing their off-target effects. Further,by modulating the molecular weight of HA in the particle matrix allowsfor tuning of the enzymatic degradation of the particles and thuscontrols the rate of antibiotic release; a property difficult torecapitulate with standard synthetic polymers. Together, this allows fortherapeutic concentrations of the drug to persist at the disease sitefor long periods of time and provide lasting anti-TB therapy. Inaddition to its influence on drug release, binding of HA by CD44receptors expressed on the surface of macrophages leads to rapid andefficient uptake of drug-loaded microgels into infected immune cells(Aruffo, et al., Cell 1990, 61 (7), 1303-1313; Kamat, et al., Bioconj.Chem. 2010, 21 (11), 2128-2135). Degradation of the particles within themacrophage phagolysosome, which also contains hyaluronidases (Goggins,et al., J. Histochem. Cytochem. 1968, 16 (11), 688-692), ultimatelyallows for intracellular release of loaded AMPs and antibiotics to clearMTb protected within these infected cellular hosts. Additionally,incorporation of these ‘carrier’ macrophages into granulomas permitsdelivery of AMPs and antibiotics to MTb protected within this nicheenvironment (FIG. 3).

In addition to standard TB antibiotics, the present invention relates tothe delivery of a new class of recently-discovered anti-TB drugcandidates that inhibit the trans-translation pathway to kill bothgrowing MTb and non-replicating persister cells (Ramadoss, et al., Proc.Nat. Acad. Sci. U.S.A. 2013, 110 (25), 10282-10287). This pathway is agood target for antibiotic development as it is required for MTb growth(Keiler, Nat. Rev. Micro. 2015, 13 (5), 285-297), is not present inanimals so specific inhibitors may not have side effects, and it has notbeen targeted for drug development in the past. However, these compoundsare limited by poor aqueous solubility and thus serve as idealcandidates to demonstrate the ability of microgels to improve drugformulation and delivery.

In summation, drug-loaded AMP microgels represent a transformative toolfor TB therapy with potential to elicit potent antimycobacterialactivity towards drug-resistant MTb, in both the replicative and latentstate, while employing an extended drug release strategy to minimizedosing frequency for TB patient.

Materials and Methods

An electrospray synthesis process has been developed that affords rapidand high-yielding production of AMP microgels (FIG. 4A; FIG. 6).Antimicrobial peptides having activity against mycobacteria, in additionto other bacterial strains, were identified. These peptides are given inTables 1, 2, and 3. Though the experimental AMPs have C-terminal amidogroups due to chemical synthesis, it is unlikely that the C-terminalamido group has a pronounced effect on the antimycobacterial effect ofthe peptides. AMPs terminated with a free carboxylate or with some otherfunctionality will likely retain their antimycobacterial properties.Microgels are prepared by spraying a 2 wt % solution of HA (100 kDa)into an aqueous bath containing 1 mg/mL of a cationic AMP with potentanti-MTb activity. Peptides were synthesized and purified using standardsolid-phase techniques, with purity >95% as indicated by LC-MS.Importantly, this synthetic approach produced gram quantities of theparticles, referred to as TB1 microgels, in <1 hour and at low cost(<$50/gram). Following purification, dynamic light scattering indicatedTB1 particles are approximately 1.5 μm in diameter (FIG. 4B). Althoughno consensus has been reached, it is generally regarded that inhalablemicroparticles 1-5 μm in size are ideally suited to distribute to thelower respiratory tract and alveolar epithelium, due to airflowconvection and gravitational effects (Kleinstreuer, et al., Annu. Rev.Biomed. Eng. 2008, 10, 195-220), while still being readily phagocytosedby alveolar macrophages (Lawlor, et al., Mol. Pharmaceutics 2011, 8 (4),1100-1112). Importantly, these particles have a negative surface charge(FIG. 4C), suggesting they should not be prematurely adsorbed to theupper airway during inhalation. Electron microscopy reveals an usual‘ruffled’ surface morphology of the particles (FIG. 4D), which suggestsa large surface area available to engage bacterial pathogens and elicitcontact-dependent lysis.

TABLE 1 Exemplary AMP sequences with anti-MTb activity. IC₉₀ (μM)^(c)En- Formal M. tuber- M. try Sequence^(a) Charge^(b) culosis smegmatisSI₉₀ ^(d) 1 WKWLKKWIK-NH₂ +5 1.1  1.9 21.3 (SEQ ID NO: 55) 2ILRWKWRWWRWRR-NH₂ +7 2.4  2.4 21.3 (SEQ ID NO: 51) 3 KRWWKWWRR-NH₂ +63.1  4.9 51.2 (SEQ ID NO: 53) 4 RRWWRWVVW-NH₂ +4 5.6 11.4 32.0(SEQ ID NO: 54) 5 KRWHWWRRHWVVW-NH₂ +7 see Table 3 N/D^(e)(SEQ ID NO: 52) ^(a)All sequences are prepared synthetically withamidated C-terminus.; ^(b)Peptide formal charge including the N-terminalamine.; ^(c)Minimum concentration inhibiting 90% culture growth.;^(d)Selectivity index at IC₉₀ against the THP-1 human macrophage controlcell line; ^(e)N/D = not determined

TABLE 2 AMPs, number of AA residues, and formal charges Formal Sequence# AA Charge AMP₁ WKWLKKWIK-NH₂  9 +5 (SEQ ID NO: 55) AMP₂ KRWWKWWRR-NH₂ 9 +6 (SEQ ID NO: 53) AMP₃ RRWWRWVVW-NH₂  9 +4 (SEQ ID NO: 54) SMAS-1KRWHWWRRHWVVW-NH₂ 13 +7 (SEQ ID NO: 52)

TABLE 3 MIC values for AMPs listed in Table 2. MIC (μM) Gram Strain AMP₁AMP₂ AMP₃ SMAS-1 + MR S. aureus >80 80 20 >80 + MS S. aureus >80 8020 >80 + B. anthrax 10 80 20 >80 − P. aeruginosa 10 20 80 50 − A.baumannii 25 80 10 50 − S. enterica 40 80 80 80 − H. influenzae 80 80 4080 +/− M. smegmatis 20 20 20 5 +/− M. tuberculosis 20 10 20 3

Three additional microgel formulations prepared using AMPs with variedcomposition, formal charge and hydrophobicity are synthesized and theirantibacterial activity tested. (Table 1, Entries 2-4). Thus, theflexibility of the electrospray synthetic approach is tested, thephysiochemical properties of the peptide cross-linker that affordscompetent microgels is identified, and a small library of particles withwhich to screen for anti-MTb activity and specificity is provided. AMPsare synthesized and purified using known methods and instrumentation(Medina, et al., Biomaterials 2015, 53, 545-553; Medina, and Schneider,J. Controlled Release 2015, 209, 317-326; Smith, et al., Nat. Nano.2016, 11 (1), 95-102; Medina, et al., Angew. Chem. Int. Ed. 2016, 55(10), 3369-3372; Ishikawa, et al., Cell Chem. Biol. 2017, 24 (2),149-158). Peptides are used at >95% purity (as assessed by LC-MS). LikeTable 1, the AMPs listed in Table 2 display potent lytic activitytowards MTb, as indicated by low micromolar IC₉₀ (inhibitoryconcentration of 90% culture growth) values (Ramón-Garcia, et al.,Antimicrob. Agents Chemother. 2013, 57 (5), 2295-2303), and M.smegmatis, a non-pathogenic strain used as a model for MTb. Importantly,these sequences are ˜20-50 times more selective in their lytic actiontowards MTb over a human macrophage control cell line (expressed asselectivity index, SI, in Table 1). This is significant as manyantibacterial peptides have narrow therapeutic indexes, and can causemembrane disruption and necrosis of healthy mammalian cells at elevatedconcentrations (Fjell, et al., Nat. Rev. Drug Discov. 2012, 11 (1),37-51). This suggests that microgels prepared from these AMPs arecapable of eliciting potent antibacterial activity with minimaloff-target effects.

Using optimized electrospray conditions (24 kV, 2 wt % of 100 kDa HA),the ability of these AMPs to form competent microgels at bathconcentrations of 0.5-3 mg/mL of the peptide is screened. Followingpurification by dialysis and lyophilization, the size and surface chargeof the particles is measured by dynamic light scattering and zetapotential analysis, respectively. Scanning electron microscopy isperformed on select formulations to confirm particle size and visualizesurface topography. Formulations that afford 1-5 μm particles, and whichpossess a neutral or negative surface charge, are tested forantimycobacterial activity and specificity against healthy human controlcell lines.

For growth inhibition studies, non-virulent MTb (H37Ra) and M. smegmatisare incubated with 0.001-10 mg/mL TB1-TB4 microgels for 24-72 hours(FIG. 7). At specific time points, antibacterial activity is evaluatedby measuring bacterial load via optical density (OD600) ofmicrogel-treated samples relative to untreated cultures or those lysedwith Triton X-100 as negative and positive controls, respectively. Inseparate experiments, MTb cells are incubated for 24 hours with selectmicrogels at their IC₉₀, and scanning electron microscopy is performedto visualize membrane disruption. The non-specific toxicity of selectedformulations is tested as a function of particle concentration towardshuman THP-1 macrophages and A549 epithelial cells as controls. THP-1 isa human monocyte line that differentiates into macrophages upontreatment with phorbol 12-myristate 13-acetate (PMA) (Auwerx,Experientia 1991, 47 (1), 22-31). A549 is a lung cancer cell line thathas been previously used as a surrogate for pulmonary epithelium(Crabbé, et al., Sci. Rep. 2017, 7, 43321; Carterson, et al., Infectionand Immunity 2005, 73 (2), 1129-1140). Selectivity indices of theformulations, calculated as IC_(90(control))/IC_(90(MTb)), is determinedfor each control cell line and candidate microgels with SI's≥20 arechosen for further study.

In separate experiments, fluorescent confocal microscopy is used todirectly observe binding of selected microgel formulations to thesurface of MTb and evaluate contact-dependent lysis.Fluorescently-labeled AMPs are prepared using previously developedprotocols (Medina, and Schneider, J. Controlled Release 2015, 209,317-326; Medina, et al., Angew. Chem. Int. Ed. 2016, 55 (10),3369-3372), the labeled peptides (10-50 μg/mL) are doped into theelectrospray bath solution containing unlabeled AMPs. The resultingfluorescent microgels are incubated with H37Ra MTb at their IC₉₀ for0.5-12 hours, followed by counterstaining treated cells with DAPI tovisualize and propidium iodide (PI) to indicate membrane disruption.These experiments allow for the visual determination of the relativecontribution of direct contact-dependent lysis, as indicated byco-localization of fluorescent particles and PI positive cells, or lossof particle integrity leading to release of free AMPs to MTb cells, onthe overall antibacterial activity of selected microgels.

The formulation of AMP microgels is optimized to afford micron-sizedparticles with potent anti-TB activity and minimal off-target toxicity.Formulations that meet the SI≥20 criteria are advanced for drug loadingand release assays. The preliminary formulation conditions affordparticles within a size range of 1-5 μm. Confirmation that AMPs aredisplayed at the microgel surface is determined by performing confocalmicroscopy on fluorescent formulations. In some cases, the HA matrix ispartially cross-linked using polylysine, a commercially availablebiocompatible polycation. Polylysine limits the diffusion of AMPS intothe particle core and thus promotes their preferential display at thesurface. In some cases, the AMPS are chemically conjugated to thesurface of pre-formed microgels to further enhance antibacterialresponses. All physiochemical characterization and in vitro studies areperformed at n≥3, with three internal replicates per experiment.Statistical significance between conditions is evaluated using un-pairedStudent's t-test assuming unequal variance (p<0.05).

Experiment 2: Combinatorial Activity of Drug-Loaded Microgels

Research has shown that the trans-translation pathway is essential forMTb growth in culture (Keiler, Nat. Rev. Micro. 2015, 13 (5), 285-297).Using bacterial reporter systems and biochemical assays, it has beendemonstrated that select oxadiazole compounds inhibit trans-translationbut not translation (Ramadoss, et al., Proc. Nat. Acad. Sci. U.S.A.2013, 110 (25), 10282-10287). High-throughput screening of >660,000compounds identified a lead agent, KKL-35, with potent activity (IC<1.6μg/mL) towards growing MTb (FIG. 5A) and dormant persister cells (FIG.5B), while being non-toxic towards human HeLa cells (IC>500 μg/mL, notshown).

In parallel with the formulation experiments in Aim 1, the loading anddelivery of the approved TB antibiotic rifampicin (RIF), as well as theinvestigational trans-translation inhibitor KKL-35, from microgels isexamined as individual agents or co-loaded into the same particle. Ofnote, although microgels may be able to deliver many different classesof antibiotics, RIF was selected for initial studies as it is afirst-line TB drug that is challenged by low water solubility anddose-limiting side effects (Laurenzi, et al., Infectious Disorders-DrugTargets 2007, 7 (2), 105-119), thus making it an ideal model with whichto test the drug formulation and delivery capabilities of microgels.Loading is performed by dissolving each compound in the HA electrospraysolution at its maximum water soluble concentration (10 mg/mL for RIF(R3501, S. P. N., RIFAMPICIN. In Product Information Sheet,Sigma-Aldrich: 1997), 500 μg/mL for KKL-35), leading to directencapsulation of the drug within microgels following AMP cross-linking.To evaluate the influence of HA molecular weight on enzyme-mediated drugrelease from microgels, drug-loaded particles are formed usingcommercially available HA of molecular weight 10 kDa, 100 kDa and 1 MDa.The length of HA is a particularly important parameter as high molecularweight variants not only undergo slower enzymatic degradation, but arealso known to inhibit pulmonary inflammation and epithelial destructionduring bacterial infection (Cyphert, et al., Int. J. Cell Biol. 2015,2015, 8). At any rate, drug loading efficiency is established bypelleting a known mass of freshly prepared microgel particles viacentrifugation (10,000 rpm for 5 min), and quantifying the concentrationof non-encapsulated drug remaining in the supernatant via HPLC. The RIFloading is determined by measuring the absorbance of purified particlesat the drug's characteristic UV maxima (λ_(max)=500 nm; Pati, et al.,Sci. Rep. 2016, 6, 24184). Loading results are expressed as the weightof drug encapsulated per unit mass of the microparticles, with an idealload of ≥40 μg/mg. This threshold is based on previous experimentsdemonstrating that a similar RIF loading into PLGA microspheres producedeffective anti-TB responses in vitro and in vivo (O'Hara and Hickey,Pharm. Res. 2000, 17 (8), 955-961; Suarez, et al., Pharm. Res. 2001, 18(9), 1315-1319).

Drug release from microgels in the absence or presence ofphysiologically relevant concentrations of hyaluronidase enzymes HYAL1and 2 (50 μg/mL) (Sahu, Biochem. Med. 1981, 25 (1), 56-61) is measuredas a function of HA molecular weight and incubation time (0-72 hours).At selected time points after enzyme addition, samples are centrifugedto pellet intact particles and the supernatant assayed by LC-MS.Concentration of released drug is determined from LC peak areas relativeto calibrations, and results are normalized to initial loadedconcentration to calculate percentage cumulative release. Tandem MSmonitors potential decomposition of the antibiotics duringenzyme-mediated particle degradation and release. Formulations thatretain the drug when incubated in blank buffered control solutions, butallow for sustained multi-day release of the cargo in the presence ofhyaluronidase, are carried forward for in vitro assays. Collectively,these studies identify the design criteria that governs drug releasefrom AMP microgels, and allow for optimization of particle formulationsto achieve long-term delivery of therapeutically relevant drugconcentrations to physiologic solutions.

The therapeutic activity of drug-loaded microgels towards growing andnon-replicating dormant MTb is evaluated through a series of aerobic andanaerobic cell-based assays, respectively. Single or dual drug-loadedformulations are added to aerobic MTb cultures (H37Ra) seeded intomulti-well plates, and bacterial growth is evaluated as a function ofparticle concentration (0.001-10 mg/mL) and incubation time (1-5 days).Similar experiments in non-proliferating persister cells are performedusing the anaerobic ‘Wayne’ model, in which non-replicating but viableMTb cultures are generated by gradual oxygen consumption in a sealedglass vial. Microgels suspended in buffer are then introduced to thecultures anaerobically via a septum, followed by stirring of thecultures for 3 days and viable bacteria quantified by plating. For bothexperiments, cultures left untreated, or lysed using Triton X-100, serveas negative and positive controls, respectively. Results are compared toMTb incubated with blank AMP microgels, or an equivalent concentrationof the free antibiotic, to identify potential synergy between AMPS andsmall molecule drugs. Microgels with activity equal to, or greater than,the free antibiotic control after a single dose are subjected toscreening against virulent and drug-resistant MTb through the NIAID TBresearch program.

These studies optimize antibiotic combination and particle compositionto afford drug-loaded microgels with sufficient potency (IC₉₀<50 μg/mL;threshold based on limitations of material yield and lung volume). Thesestudies demonstrate that drug-loaded AMP microgels lead to more potentand sustained anti-TB activity than the standard of care of a bolus doseof antibiotics. In cases of poor drug solubility in the aqueous HAelectrospray solution and low loading efficiencies, DMSO is added (up to5% v/v) until loading above the objective threshold (≥40 μg/mg for RIF)is achieved. For RIF in particular, a lactose blend is employed (90:10RIF:lactose) previously shown to enhance RIF loading into PLGAmicrospheres (O'Hara and Hickey, Pharm. Res. 2000, 17 (8), 955-961). Insome cases, changing HA molecular weight to modulate the enzymaticdegradation of microgels is not sufficient on its own to effectivelycontrol the rate of drug release. In cases where release is too slow, asindicated by sub-therapeutic concentrations of drug released after 3days, the porosity of the microgel matrix is increased by reducing theconcentration of the AMP cross-linker. Otherwise, salt leeching isemployed to increase the porosity of cross-linked materials (Annabi, etal., Tissue Eng. B, Rev. 2010, 16 (4), 371-383).⁴⁴ In cases where drugrelease is too rapid, such that complete release occurs within hours,drug diffusion is slowed by employing high AMP concentrations toincrease particle matrix density, or by including polylysine as aco-crosslinker. To obtain rigorous and reproducible results, drugrelease and in vitro studies are performed at n≥3, with three internalreplicates per experiment. Statistical significance is established viaone-way ANOVA (p<0.05).

Experiment 3: Microgel Sterilization of Infected Macrophages and CargoDelivery into Granulomas

The incubation time required for microgel phagocytosis by macrophages isoptimized using live-cell fluorescence microscopy. Here, differentiatedTHP-1 cells are incubated for 0.5-6 hours with rhodamine-labeled AMPmicrogels (prepared as described elsewhere herein) at a 1:25cell:particle ratio (Pacheco, et al., PLoS One 2013, 8 (4), e60989).Treated macrophages are counterstained with calcein-AM and DAPI tovisualize cells and confirm intracellular uptake of rhodamine-labeledparticles. The ability of drug-loaded microgels to clear MTb frominfected macrophages is tested using an in vitro co-culture infectionmodel (Estrella, et al., Front. Microbiol. 2011, 2, 67). Briefly,differentiated THP-1 cells are incubated with MTb (H37Ra) at amultiplicity of infection of 10 for 24 hours to allow for microbeuptake. Cells are then washed to remove non-phagocytosed bacteria,seeded into multi-well plates and treated with drug-loaded microgels at0.1-10 mg/mL concentrations to initiate particle phagocytosis. After thepredetermined treatment period, cells are washed to removeun-internalized particles and incubated for an additional 5 days inblank medium. Changes in the bacterial load of treated macrophages arequantified by directly counting the number of bacilli per cell. To thisend, treated cells are fixed, stained with the mycobacterium specificmarker auramine-rhodamine, and counter-stained with DAPI to visualizeTHP-1 cells before performing fluorescent microscopy. In parallelexperiments, treated macrophages are lysed, the lysate plated onto agar,and the samples incubated for 2 to 3 weeks to determine theintracellular CFU load. For both of these studies, results are comparedto control samples of untreated infected macrophages, cells incubatedwith blank microgels, or those given free antibiotic(s), to assess theenhanced efficacy of drug-loaded microgels relative to free drug.

The delivery of antibiotics and AMPS into granuloma tissue is testedusing a modified in vitro model (Sarathy, et al., JoVE 2017, (123),e55559). In brief, PMA-activated THP-1 cells are incubated with 0.4 mMoleic acid overnight to form foamy macrophages. These cells are thenpelleted, lysed and denatured at 75° C. to produce a cell amalgam,‘caseum’, that mimics the composition and architecture of necrotic TBlesions. The caseum pellets are sectioned into ˜11 mm discs and placedinto 24 well plates. To initiate treatment, cell culture mediacontaining viable PMA-activated THP-1 cells and 0.1-10 mg/mL of selecteddrug-loaded microgels, or an equivalent concentration of free drug as acontrol, is added to each well and incubated for 5 days. This procedureallows for uptake of microgels into macrophages and their subsequentincorporation into the necrotic granuloma lesion. Following treatment,the caseum samples are washed, embedded in paraffin and sectioned usinga hand microtome to allow for analysis via time-of-flight secondary ionmass spectrometry (ToF-SIMS). Importantly, ToF-SIMS can detect thepresence of defined molecular weight species across a sample surface,and thus allows for the construction of a 3D map to visualize thespatial localization of delivered AMPS and antibiotics within the modeltissue. Results are compared to control samples treated with anequivalent concentration of the free antibiotic(s).

These studies test the capacity of drug-loaded microgels to clear MTbwithin infected macrophages, and assesses their ability to enhance thepenetrance and delivery of therapeutic cargo within granuloma tissue.These studies allow for the translation of selected microgelformulations into preclinical animal models. Uptake of microgels intoTHP-1 cells is not a significant problem, as the rapid phagocytosis ofHA microparticles into macrophages is known (Kamat, et al., Bioconj.Chem. 2010, 21 (11), 2128-2135). In cases where poor MTb clearance isobserved in the co-culture infection assay, multi-drug cocktailstypically used in the clinic (e.g. RIF, isoniazid, pyrazinamide andethambutol) are encapsulated and delivered. Ambiguous ToF-SIMS analysesof the in vitro model granuloma tissue are further confirmed using rapidequilibration dialysis (RED) experiments (Sarathy, et al., JoVE 2017,(123), e55559). THP-1 cell pellets are homogenized, mixed withdrug-loaded microgels and placed within the donor chambers of REDinserts. Blank buffer is placed in the cognate receive chamber, andsamples incubated for 4 hours. Subsequent LC-MS analysis of the donorand receiver chamber allow for the quantification of the amount ofantibiotic retained within the caseum tissue treated with drug-loadedmicrogels relative to control samples incubated with the free drug. Toobtain rigorous and reproducible results, in vitro studies are performedat n≥3, with three internal replicates per experiment. Statisticalsignificance is established via unpaired Student's t-test assumingunequal variance (p<0.05).

Experiment 4: Biohybrid Peptide Nanogels that Augment the Utility ofBioactive Cargo

Bioresponsive nano-carriers formed from readily available organicbuilding blocks, selected from the list of generally recognized as safe(GRAS) compounds by the U.S. Food and Drug Administration, have beendeveloped. A small library of GRAS components were systematicallyscreened under electrospray ionization conditions to identifycombinations that form physically cross-linked nanomaterials in highyield and low cost. Table 4 shows the qualitative results of thislibrary screening process. During these studies, it was discovered thatspraying hyaluronic acid (HA) nanodroplets into a bath ofϵ-poly-L-lysine (PLL) templated the assembly of the polycation intoelectrostatically complexed ‘gel-like’ nanoparticles, herein referred toas a nanogel (FIG. 8). This facile aqueous approach eliminates the needfor toxic co-solvents common in the synthesis of many nanoparticlescaffolds, and yields gram-quantities of peptide nanogels in <1 hour andat low cost. Small molecules or proteins present during nanogel assemblyare readily encapsulated within the particle matrix and can becontrollably released upon swelling of the carrier in physiologicsolutions. A series of biophysical and cell-based assays demonstratethat peptide nanogels augment and enhance the utility of deliveredcargoes; notably improving the potency of loaded antibiotics andchemotherapeutics, as well successfully delivering otherwisemembrane-impermeable proteins to the cytoplasm of treated cells. Peptidenanogels represent a novel class of bioresponsive carriers that canimprove the therapeutic and diagnostic utility of bioactive payloadschallenged by poor cell permeability, low bioavailability and limitedsolubility.

TABLE 4 Qualitative studies of GRAS components for nanoparticleformation Anion Hyaluronic Acid (HA) Alginate (Alg) CationPolyethylimine Film Large particles, (PEI) abnormal, agglomerate/gelCalcium No result Microparticles, (Ca²⁺) spherical, uniformpoly-L-lysine Nanoparticles, uniform, Large agglomerate/gel (PLL) turbidsolution

Materials and Methods

Hyaluronic Acid (MW: 100 kDa) was purchased from Lifecore Biomedical(Chaska, Minn.). 0.1% (w/v) poly-L-lysine (x=400) was purchased fromAlamanda Polymers (Huntsville, Ala.). Vancomycin hydrochloride and MTTpowder were purchased from Chem-Impex (Wood Dale, Ill.). Greenfluorescent protein was provided by the Schneider Group (NCI/NIH;Frederick, Md.). Doxorubicin hydrochloride was purchased from OakwoodChemical (Estill, S.C.). Formic acid, LC/MS grade acetonitrile, Cationadjusted Mueller-Hinton broth and Glutamax were purchased from ThermoFisher Scientific (Waltham, Mass.). 300 kDa MWCO dialysis tubing waspurchased from Spectrum (Rancho Dominguez, Calif.). P. aeruginosa and S.aureus were provided by the Chroneos Group (Hershey Medical Center;Hershey, Pa.). E. coli was provided by Pak Kin Wang's Group (Dept. ofBiomedical Engineering; University Park, Pa.). A. baumannii and S.enterica provided by the Keiler Group (Dept. of Biochemistry andMolecular Biology; University Park, Pa.). NL-20 cells were provided byMatthew Taylor's group (Hershey Medical Center; Hershey, Pa.). RPMI 1640culture medium was purchased from Lonza (Basel, Switzerland). FetalBovine Serum (FBS), L-glutamine (L-Gln), Trypsin, Phosphate BufferedSaline (PBS) and Dulbecco's Modified Eagle Medium (DMEM) were purchasedfrom Corning (Corning, N.Y.). Gentamicin and Ham's F12 medium werepurchased from VWR (Radnor, Pa.). HUVEC (ATCC PCS-100-010), VascularCell Basal Medium and the Endothelial Growth Cell Kit—VEGF werepurchased from ATCC. EmbryoMax Ultrapure Water with 0.1% Gelatin, MEMNon-Essental amino acid solution 100×, D-(+)-Glucose, Recombinant humaninsulin, Human Plasma Transferrin Apo-, Hudrocortisone, and Epidermalgrowth factor were purchased from Sigma Aldrich (St. Louis, Mo.).

The PLL bath solution was made by 10-fold dilution of stock solutioninto autoclaved DI water. The solution was filtered at 0.2 μm(polyethylene sulfone, VWR; Radnor, Pa.) and a final volume of 30 mL wasachieved. The HA spray solution concentration was determined by N:Pratio, the molar ratio of negative to positive charged units (assumingall are concurrently ionized), with PLL concentration remainingconstant. The HA was dissolved in autoclaved DI water at 37° C. and thesolution was filtered at 0.2 μm by polyethylene sulfone with a finalvolume of 3 mL. The HA solution was loaded into a 5-mL syringe andattached to a 0.5 inch 28-gauge needle (Hamilton; Reno, Nev.). A loadwas applied from a grounded high voltage power supply (230-30R,Spellman; Hauppauge, N.Y.). The PLL bath was set in a glass petri dishwith a submerged common ground wire. HA was sprayed into the bath at 0.1mL/min (Pump 11 Elite, Harvard Apparatus; Holliston, Mass.) to a totalspray volume of 2 mL. The resulting particle solution was incubated at37° C. for 1 hour. Then the particles were centrifuged (Centrifuge 5430R, Eppendorf; Hamburg, Germany) at 10,000×G at 25° C. for 30 minutes andwashed once with autoclaved DI water. The washed particles were slowfrozen in isopropanol to −80° C. and lyophilized (FreeZone 4.5,Labconco; Kansas City, Mo.) overnight if storage or later experimentsrequired.

After the preparation of nanogels was complete, samples werecharacterized by dynamic light scattering (DLS) and zeta potential(Zetasizer Nano ZS, Malvern; Malvern, United Kingdom). Scanning electronmicroscopy (SEM; NanoSEM 630, FEI; Hillsboro, Oreg.) was employed toconfirm DLS results. Nanogels in suspension were allowed to air drydirectly on specimen stubs before iridium (Ir) sputter treatment.

Following the centrifugation washing, nanogels were resuspended inDulbecco's Modified Eagle's Medium (DMEM). DLS measurements were takenimmediately after resuspension, and at predetermined timepoints, untilno comprehensible signal was recorded. Particle suspensions wereincubated at 37° C. for the duration of the experiment, and mixed byinversion before each measurement.

Vancomycin hydrochloride was dissolved at 75 mg/mL in an HA solutionprepared at an N:P of 10. The 0.01% PLL bath was made and the Vanco-HAsolution was electrosprayed into the bath following the method laid outin the previous section. After centrifugation, the loaded particles(Vanco-BNCs) were resuspended in DI water. Loading was characterized bysonicating Vanco-BNCs and measuring absorbance at 280 nm onreverse-phase HPLC using a C18 analytical column from Phenomenex(Torrance, Calif.). HPLC solvents consisted of solvent A (0.1% formicacid in water) and solvent B (0.1% formic acid in 9:1acetonitrile/water) with a linear gradient of 0-100% solvent B over 25min.

Green Fluorescent Protein (GFP) was dissolved at 0.1 mg/mL in the 0.01%PLL bath solution. The HA solution was prepared at an N:P of 10,electrosprayed following the method described elsewhere herein. Theloaded particles (GFP-BNCs) were washed following the samecentrifugation method used for unloaded particles. The resulting pelletwas resuspended in DI water, and filtered at 0.2 μm by centrifugefilter. Loading was characterized by measuring fluorescence (λ_(ex)=470nm, λ_(em)=515 nm) of 100 μL in a 96 well plate on a microplate reader(Cytation 3, BioTek; Winooski, Vt.).

Unloaded BNCs were resuspended in DI water to a concentration of 2mg/mL. Doxorubicin hydrochloride was weighed out to achieve a 1:1particle:Dox mass ratio, solubilized in an equal volume of DI water andstirred overnight. The loaded particles (Dox-BNCs) were washed followingthe same centrifugation method used for unloaded particles. Theresulting pellet was resuspended in DI water, and filtered at 0.2 μm bycentrifuge filter. Loading was characterized by measuring fluorescence(λ_(ex)=480 nm, λ_(em)=570 nm) of 100 μL in a 96 well plate on amicroplate reader.

Loaded BNCs in DI water were injected into 300 kDa MWCO dialysis tubing.The tubing was submerged in a 30× (by volume) bath of PBS. Atpredetermined time points, multiple samples (n≥3) were collected frommultiple locations throughout the bath. These samples were pooled andrelease was characterized in the same method used for assessing initialloading. Before each measurement, volume was adjusted to that of theinitial bath to ensure concentration consistency.

2× Dilutions of treatments in broth were plated into a U-bottom 96 wellplate. Overnight cultures of Pseudomonas aeruginosa, Escherichia coli,Acinetobacter baumannii, Salmonella enterica and Staphylococcus aureuswere incubated in cation adjusted Mueller-Hinton broth as advised byCLSI. Bacteria were diluted to an OD₆₀₀ of 0.002 and plated in a 1:1volume ratio with the treatments. These cultures were incubatedovernight and MIC values were assessed by visual evaluation of growthinhibition when compared to untreated samples.

Cell suspensions of A549, NCI/ADR-RES, HUVEC and NL-20 cells were platedinto a 96-well plate (2×10³ cells/well) and incubated for one day incomplete culture medium. A549 and NCI/ADR-RES cells were cultured inRPMI-1640 supplemented with 10% FBS, 1% L-Gln, and 0.1% gentamicin at37° C. and 5% CO₂. HUVEC were cultured in Vascular Cell Basal Mediumsupplemented as described on ATCC in flasks coated with gelatin (0.1%for 15 minutes at 37° C.) under the same conditions. NL-20 cells werecultured in Ham's F12 as described on ATCC under the same conditions.The cells were treated after removing the previous complete mediumsupernatant and incubated for 48 hours before removing the treatmentsupernatant. Cell viability was assessed by adding 0.5 mg/mL MTTsolution in complete culture medium into each well. After standardincubation for 2 hours, the cells were read with a microplate reader at540 nm. The resulting data was analyzed using nonlinear regression ofsemi log data as performed by GraphPad Prism.

Cell suspensions of A549 cells were plated into a 24-well plate (5×10⁴cells/well) with a sterile cover slip and incubated for one day incomplete culture medium at 37° C. and 5% CO₂. The cells were treatedafter removing the previous complete medium supernatant and incubatedfor 72 hours before removing the treatment supernatant. Cells wererinsed with fresh media to remove excess treatment and fixed in 4%paraformaldehyde for 15 minutes. Hoechst stain was applied at 2 μg/mLfor 15 minutes and washed with PBS. Fixed stained cultures were storedat 4° C. until imaging by confocal microscopy (FV1000, Olympus;Shinjuku, Japan).

The results of the experiments will now be described

Exploiting the assembly of bio-sourced and biodegradable GRAS compoundsis an attractive strategy in the design of environmentally-sensitive, or‘green’, engineered nanomaterials. These components are ofteninexpensive, readily available in bulk, and present a lower barrier toregulatory approval compared to synthetic analogues. With this in mind,a library of commercially available GRAS compounds was tested toidentify suitable combinations under which bioresponsive nanoparticlescould be prepared via electrospray ionization. Electrospraying is aprocedure by which a high voltage is applied to a sample solution asit's passed through a capillary tip. Coulombic repulsion within theejected solution generates a fine nanodroplet mist that is collected ina bath solution containing the complimentary cross-linker. This facilesynthesis method provides a convenient means to rapidly screen differentpermutations of oppositely charged molecules to assess their potentialto form competent nanomaterials in bulk. During these studies it wasobserved that many of the electrosprayed GRAS mixtures generated surfacefilms, fibrous amalgams or large amorphous aggregates. However, oneparticular combination of spraying a solution of hyaluronic acid (HA)into a bath of poly-L-lysine (PLL) rapidly generated a monodispersesuspension of ˜120 nm particles (FIG. 9A), which remained colloidallystable in bulk solution (FIG. 9B). Interestingly, the final size ofassembled peptide nanogels appeared to be insensitive to changes in thevoltage applied to the metal capillary tip during electrospray synthesis(FIG. 10A).

To better understand the relative distribution of the HA and PLLcomponents within the nanogel matrix zeta potential measurements wereemployed to probe the particle's surface composition. FIG. 9C shows thatnanogels possess a highly electronegative surface (−35 mV), indicatingthat the particles are likely comprised of an anionic HA shell thatsurrounds a PLL-rich core. Next, we tested how the density ofelectrostatic cross-links in the particle network impacts the size ofassembled nanogels. This was done by adjusting the concentration of HAin the spray to vary the stoichiometric ratio of negative (N; COO⁻ ofHA) to positive (P; NH₃ ⁺ of PLL) groups available to assemble the finalparticle matrix. We found that varying the N:P ratio from 1 to 15 formedpeptide nanogels of uniform size (FIG. 9D, FIG. 10B). Further, weobserved a similar surface charge for particles prepared at an N:P of 5and 10, suggesting that the core-shell architecture of nanogels is notinfluenced by the relative number of cross-links that comprise thematrix (FIG. 10C). Attempts to form nanogels at N:P ratios <1 resultedin instable particles that could not be accurately measured by DLS orzeta potential analysis. Collectively, these results indicate that thecross-linking density of the nanogel network can be carefully tuned,independent of the final particle size, and in turn may allow forcontrol over the swelling behavior of the particles in physiologicsolutions.

In order to test the above assertion, nanogels formulated with differentN:P ratios were suspended in 37° C. cell culture media and measured thechange in particle size as a function of time. FIG. 11A shows that therate of nanogel swelling is inversely related to the amount of HA in thenetwork. Further, the time to particle failure, or the point at whichthe network can no longer sustain a competent gel, increased from 1-72hours as the N:P ratio was raised 1 to 15. Interestingly, nearly all ofthe tested nanogel compositions reached a maximum swollen diameter of˜300 nm, representing an approximate 2.5-3.0 fold increase in size,before complete decomposition (as indicated by disappearance of the DLSsignal). Conversely, nanogels suspended in deionized water remainedstable and showed no time-dependent change in size (FIG. 10D). Scanningelectron microscopy (SEM) performed on particles before and afterswelling demonstrates that peptide nanogels possess a smooth surfacetopology that is maintained during their expansion, without apparentfragmentation or particle agglomeration (FIG. 11B). Together, theseresults suggest that disruption of the nanogel electrostatic matrix byinfiltrating salt ions leads to bulk expansion of the biopolymernetwork. The matrix continues to swell until a critical entanglementthreshold is reached and particle integrity is lost. Importantly, thetime to nanogel failure is linearly dependent with the N:P ratio usedduring their synthesis (FIG. 11C). This suggests that nanogeldecomposition can be carefully controlled with high temporal resolutionto afford precise release of encapsulated cargo.

To demonstrate the utility of nanogels for drug delivery applications, aseries of release experiments were perfomed following the encapsulationof three different molecular cargoes: the model biomacromolecule greenfluorescent protein (GFP), the small molecule chemotherapeuticDoxorubicin (Dox), and the antibiotic Vancomycin (VAN). Nanogels formedfrom an N:P of 10 were used in these studies, as these particles couldbe readily prepared in bulk and showed a multi-day swelling profileamenable to sustained drug delivery (FIG. 11A). Loading of the variouscargoes into the nanogel carrier was performed using three differentoptimized procedures (FIG. 12A). This included incorporation of theagent in the sprayed HA solution (VAN) or the PLL bath (GFP), leading toits direct encapsulation during particle assembly. In the case of DOX,incubating pre-assembled nanogels with the hydrophobic drug led tooptimal loading. Attempts to include DOX in the spray or bath solutionsled to amorphous precipitates during particle assembly. At any rate,these varied loading methods highlight the ability of the nanogelelectrospray synthesis procedure to be readily adapted for effectiveencapsulation of a variety of cargoes with vastly different solubilityand physiochemical properties. DLS analysis confirmed that loading ofthe different agents into the nanogel carrier did not significantlyimpact their size (FIG. 10E).

Next, physiologic release of the encapsulated cargo was assessed byloading each nanogel formulation into a dialysis cassette and suspendingit in a release media of 37° C. PBS. The concentration of loaded drug orGFP liberated to the dialysis media was then monitored as a function oftime via UV-Vis or fluorescence spectroscopy, respectively. Results inFIG. 12B show that the hydrophilic drug VAN is rapidly released from thenanogel carrier with first order kinetics, achieving 90% drug releasewithin 4 hours. DOX, on the other hand, displays a zero-order releaserate of ˜4%/hour, leading to its complete release after 48 hours.Interestingly, GFP loaded formulations showed a very different releaseprofile. The majority of encapsulated protein (>75%) was retained withinthe nanogel carrier for the first 24 hours, followed by a rapid releasephase that occurred between 24 and 72 hours. Taken together with thenanogel swelling data (FIG. 11A), this suggests that much of the loadedmacromolecular protein remains entrapped within the carrier network asit swells, and only achieves complete release upon particle disruption.

Varying the cross-linking density of the nanogel carrier, as well as thephysiochemical properties of the cargo, can be used to carefully controlthe release profile of loaded agents. To better understand how themethod of encapsulation impacts distribution of the cargo within thecarrier, and thus influences its release, zeta potential analysis wasperformed on the loaded formulations (FIG. 12C). Unloaded controlnanogels (NG) are characterized by a surface zeta potential of ˜35.3 mV,which did not significantly change when GFP is encapsulated within thecarrier (NG_(GFP)). This suggests that the protein is largelysequestered within the PLL-rich core (see schematic representation inFIG. 12D). Here, suspension of the negatively charged GFP protein, whichpossess an isoelectric point of ˜5.8, in the electrospray bath solutionlikely leads to its initial complexation with the cationic PLLcross-linker before nanogel assembly. Conversely, as VAN is contained inthe HA spray solution, the drug is entrapped within the HA nanogelcorona and thereby partially passivates its electronegative surfacecharge. This is corroborated by zeta potential measurements, which showan increase in the surface charge of VAN-loaded nanogels to ˜15.4 mV.Finally, DOX loaded formulations showed a complete neutralization ofparticle surface charge as indicated by a zeta potential of 0.6 mV. Thisis a result of the loading method employed for DOX encapsulation, inwhich pre-formed nanogels were incubated with a saturated solution ofthe drug to drive it into the particle network. At these saturatingconcentrations, it's likely that a fraction of the unloaded DOXmolecules coat the particle surface and thus neutralizes its exteriorcharge. It is worth noting that due to the small size of peptidenanogels (˜120 nm) more advanced analytical methods, such as confocalmicroscopy or TOF-SIMS, could not be applied to better assess thedistribution of loaded molecules within the particle network. The datasuggest that, in addition to nanogel N:P ratio and physiochemicalproperties of the loaded agents, the method of encapsulation can bevaried to control sub-particle localization of the cargo and henceprovide an additional degree of freedom to tune release.

Based on the favorable protein and drug release profiles from peptidenanogels, the delivery potential of the particles was assessed usingthree independent in vitro experiments. The first involved incubatingGFP-loaded nanogels with cancer cells to evaluate intracellular deliveryof the protein cargo using fluorescent confocal microscopy (FIG. 13).These studies revealed a remarkable capacity of nanogels to shuttlemembrane-impermeable GFP proteins into cells (FIG. 13A), leading toa >11 fold enhancement in intracellular fluorescence compared to cellstreated with the free protein (FIG. 13B). To investigate potentialmechanisms behind the preferential uptake of NG_(GFP), competitiveinhibition experiments were performed in which cells were co-incubatedwith GFP-loaded particles and an excess of free HA (750 ug/mL). Thesestudies evaluate the contribution of HA carbohydrates contained withinthe nanogel matrix, which bind to CD44 cell-adhesion receptors expressedon the surface of mammalian cells, towards endocytic uptake of theparticles. Images in FIG. 13A show that an excess of free HA present insolution leads to a substantial decrease in NG_(GFP) uptake, resultingin an approximate 3 fold loss of intracellular fluorescence compared tocells treated with the particle alone (FIG. 13B). Next, we co-stainedfree GFP or NG_(GFP) treated cells with fluorescently-labeledtransferrin, a marker for receptor-mediated endocytosis. The mergedfluorescent images shown in FIG. 13C indicate that NG_(GFP) treatedcells are characterized by an approximately equal fraction ofintracellular GFP delivered to the cytoplasm (green only) orco-localized with endosomes (yellow). Collectively, this data suggeststhat NG_(GFP) particles are internalized into cells via CD44receptor-mediated endocytosis, where a portion of the particles thensubsequently escape to accumulate in the cytoplasm. This suggests thatbiomacromolecules delivered by peptide nanogels into cells can betransported to the cytoplasm to avoid long-term sequestration withinendosomes and lysosomes.

Non-drug-loaded microgels prepared using antimicrobial peptides as thecationic polymer display remarkable activity against gram-negativebacteria relative to mammalian lung epithelial cell controls (FIG. 14A).In a second set of experiments, the chemotherapeutic potency of DOXdelivered from the nanogel carrier towards drug-sensitive (A549) orDOX-resistant (NCI/ADR-RES) cells was evaluated (FIG. 14B). Free DOXdemonstrates an IC₅₀ of 0.4 μg/mL (0.7 μM) and 6.5 μg/mL (12.0 μM)towards A549 and NCI/ADR-RES, respectively. Gratifyingly, delivery ofDOX via the nanogel carrier resulted in a marked increase in drugpotency towards both cell lines. For example, treatment of A549 cellswith NG_(DOX) resulted in a >10-fold enhancement in drug cytotoxicity(IC₅₀=0.03 μg/mL; 0.06 μM of equivalent drug), compared to samplestreated with free DOX. Control samples treated with empty nanogels (NG)show the un-loaded carrier is well tolerated by both cell lines.

Next, the antibacterial activity of VAN-loaded nanogels were evaluatedagainst a panel of gram-negative and gram-positive bacterial pathogens(Table 5). It is important to note that clinical use of VAN is generallyexcluded to gram-positive infections as most gram-negative pathogens areinnately resistant to the drug. This is due to the thick outer membraneof gram-negative bacteria which prevents the large glycopeptide drugfrom diffusing into the cell wall and reaching its enzymatic target. Notsurprisingly then, weak activity of VAN was observed towards fourdifferent gram-negative pathogens, leading to inhibition of bacterialgrowth only at the highest concentration tested (144 μg/mL). Conversely,the drug was >30 times more effective in killing the controlgram-positive strain Staphylococcus aureus (S. aureus).

TABLE 5 Minimum inhibitory concentration (MIC) of free VAN, VAN-loadednanogels (NG_(VAN)) or the empty nanogel carrier (NG) against a panel ofgram-negative (−) bacteria, or the model gram-positive pathogen S.aureus (+). Results are shown as the MIC of the drug, or the equivalentconcentration of drug loaded into nanogels, as well as the correspondingamount of the carrier (represented as VAN | NG carrier). MIC of VAN NGCarrier [μg/mL] P. aeruginosa (−) A. baumannii (−) S. enterica (−) E.coli (−) S. aureus (+) VAN 144* NA 144* NA 144* NA 144* NA 4.5 NANG_(VAN) 72  44 72  44 72  44 36  22 0.3  0.2 NG NA >100* NA >100*NA >100* NA >100* NA >0.3* NA = not applicable. *indicates maximumconcentration tested.

Remarkably, when VAN is loaded into the nanogel carrier, significantincreases in its potency towards both gram-negative and gram-positivebacteria were observed. For example, treatment of E. coli withVAN-loaded nanogels led to an equivalent drug MIC of 36 μg/mL, a 4-foldenhancement compared to the activity of free VAN. Similarly, NG_(VAN)killed the gram-positive S. aureus strain at a 15-times greater potencyof equivalent drug relative to bacteria treated with VAN alone. Similarto previous experiments, no toxic effects of the un-loaded nanogelcarrier (NG) towards all the tested strains were observed.

The biocompatibility of peptide nanogels was assessed in healthy humanendothelial cells and bovine red blood cells (RBCs) following a 24 hourincubation with the particles (FIG. 15). Peptide nanogels showed noovert toxicity towards human umbilical vein endothelial cells (HUVEC),as indicated by maintenance of cell viability across a range of nanogelconcentrations up to 40 μg/mL (FIG. 15A). Similarly, particles werenon-hemolytic towards bovine RBCs (FIG. 15B, note 0-2% total hemolysisfor all nanogel conditions) even when employed at concentrations ordersof magnitude greater than what was required to achieve a therapeuticresponse in the DOX (FIG. 14) and VAN (Table 5) delivery studies.Collectively, these results demonstrate that peptide nanogels are ahighly biocompatible delivery platform and suggest that parenteraladministration of the particles to vasculature will be well tolerated.

Enhancing the utility of new therapeutic and diagnostic agents viananoparticle-based delivery platforms requires materials that arechemically tractable, synthetically facile and are innatelybiocompatible. The present invention relates to a new class of peptidenanogel particle that can be rapidly prepared in high yield and purityvia electrostatic complexation of complimentary charged HA and PLLbiopolymers. Peptide nanogels represent a unique class of bioresponsivenanoparticles with tunable swelling and release profiles. Remarkably,nanogels display broad efficacy in a range of delivery applications,including successful delivery of a membrane-impermeable protein intocells, improving the potency of loaded chemotherapeutics towardsdrug-sensitive and -resistant cancer cells, and sensitization ofintractable bacterial pathogens to antibiotic.

In vitro studies suggest that peptide nanogels augment the activity ofdelivered cargo through three potential mechanisms. First, binding of HAin the carrier matrix to CD44 adhesion receptors on the cell surface mayyield a high local concentration of delivered cargo at the membrane.Second, intracellular uptake of peptide nanogels may permit moreeffective delivery of otherwise membrane-impermeable, or poorlypermeable, into cells. Third, the cationic amphiphile PLL is known toorganize with phospholipid head groups to display membranepermeabilizing effects via carpet-like mechanisms. Permeabilization ofboth plasma and endosomal membranes may be one mechanisms by which GFPis effectively delivered into cells via our nanogel carrier. Likewise,permeabilization of cancer or bacterial cell membranes by PLLincorporated within the particles may contribute to the markedenhancement in chemotherapeutic- and antibiotic-loaded formulations.Importantly, while these nano-scale materials are capable of augmentingthe activity of loaded biosensors and drugs, they are inherentlybiocompatible, non-toxic and non-hemolytic. Thus peptide nanogelsrepresent a potential theranostic platform with broad applications indrug delivery and biomedical imaging.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

We claim:
 1. A plurality of drug delivery particles, comprising: ananionic polymer matrix; and a cationic polymer; wherein the anionicpolymer matrix and cationic polymer together form drug deliveryparticles bound by electrostatic interactions; and wherein the drugdelivery particles comprise at least one biologically active agent. 2.The composition of claim 1, wherein the anionic polymer matrix comprisesan anionic polymer selected from the group consisting of alginic acid,arabic acid, polygalacturonic acid, poly(glucuronic acid), hyaluronicacid, heparin, N-acetyl heparin, carboxymethylcellulose, chondroitinsulfate, chondroitin sulfate B, chitin, O- or N-sulfochitosan,CM-dextran, dextran sulfate, and pectin.
 3. The particles of claim 1,wherein the anionic polymer matrix comprises hyaluronic acid.
 4. Theparticles of claim 1, wherein the cationic polymer is a polypeptide. 5.The particles of claim 4, wherein the cationic peptide is poly-L-lysine,6. The particles of claim 4, wherein the cationic peptide comprises asequence selected from the group consisting of ILRWKWRWWRWRR (SEQ IDNO:5), KRWHWWRRHWVVW (SEQ ID NO:11), KRWWKWWRR (SEQ ID NO:12), RRWWRWVVW(SEQ ID NO:35), and WKWLKKWIK (SEQ ID NO:46).
 7. The particles of claim1, wherein the zeta potential of the particles is negative.
 8. Theparticles of claim 1, wherein the at least one biologically active agentis selected from the group consisting of an antimycobacterial agent, anantimicrobial agent, an antiviral agent, an anticancer agent, and abiologic.
 9. The particles of claim 1, wherein the at least onebiologically active agent is selected from the group consisting ofrifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin,4-chloro-N-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)benzamide,vancomycin, and doxorubicin.
 10. The particles of claim 1, wherein thedrug delivery particles further comprise at least one pharmaceuticallyacceptable carrier.
 11. A dry powder formulation comprising theparticles of claim
 1. 12. A method of treating a mycobacterial infectionin a subject in need thereof, the method comprising the step ofadministering to the subject a formulation comprising the particles ofclaim
 1. 13. A method for the manufacture of drug delivery particles,the method comprising the steps of: providing a sample solutioncomprising at least one anionic polymer; providing a bath solutioncomprising at least one cationic polymer; electrospraying the samplesolution into the bath solution to form a drug delivery particlesolution; and isolating a plurality of drug delivery particles from thedrug delivery particle solution; wherein at least one of the samplesolution, the bath solution, or the solution of drug delivery particlesfurther comprises at least one biologically active agent.
 14. The methodof claim 13, wherein the step of isolating a plurality of drug deliveryparticles from the drug delivery particle solution comprises the step ofcentrifuging the drug delivery particle solution and removing thesupernatant.
 15. The method of claim 13, wherein the step of isolating aplurality of drug delivery particles from the drug delivery particlesolution further comprises the step of lyophilizing the drug deliveryparticles.
 16. The method of claim 13, wherein the step ofelectrospraying the sample solution into the bath solution to form adrug delivery particle solution further comprises the step of incubatingthe drug delivery solution for at least one hour at least 37° C.
 17. Themethod of claim 13, wherein the bath solution comprises the biologicallyactive agent.
 18. The method of claim 13, wherein the at least oneanionic polymer comprises hyaluronic acid.
 19. The method of claim 13,wherein the at least one cationic polymer comprises poly-L-lysine or anantimicrobial polypeptide.
 20. The method of claim 13, wherein theelectrospray voltage is between about 10 kV and about 50 kV.